Afterglow Luminescence of Lu2O3Eu Ceramics Synthesized at Different Atmospheres

抗衰老系列二科学家公布外泌体抗衰老实验结果2013年诺贝尔生理学或医学奖颁给了美国科学家罗思曼、谢克曼以及德国科学家祖德霍夫，以表彰他们发现细胞的囊泡运输调控机制。

美国抗衰老医学研究会主席Klatz博士称：“外泌体是干细胞技术的下一个发展方向”。

那么，人们面临衰老时，干细胞外泌体正在发挥着怎么样的作用？科学家们又是如何利用干细胞外泌体来对抗衰老？本期向大家介绍一项重要的外泌体抗衰老实验。

抗衰老系列二 | 科学家公布外泌体抗衰老实验结果干细胞在再生医学中具有巨大的治疗潜力，如通过分泌抗炎、抗纤维化和促血管生成活性的因子，如可溶性分子（生长因子、细胞因子）或细胞外囊泡（微粒子、外泌体）等来改善创面愈合过程。

随着研究的深入，干细胞外泌体成为备受青睐的“超级明星”。

干细胞外泌体，干细胞在生理活动过程中分泌出来的生理活性物质，是一种微小囊泡，直径大约在30-150nm。

主要功效成分包括蛋白质类物质及micRNA类核酸物质。

外泌体一旦通过胞吐作用从干细胞中释放，复杂的混合因子能作为信号分子传递给其他细胞。

干细胞外泌体因在上皮组织的增殖、迁移、再生、炎症和瘢痕控制等方面的作用，成为「无细胞的细胞治疗」工具。

细胞老化是什么除去部分的组织干细胞，构成我们身体的大部分细胞的分裂次数是有限的，正常的体细胞达到细胞寿命并不可逆地停止增殖的状态称为细胞老化。

另外，就算添加活性正常的细胞至有致癌危险的应激反应（染色体缩短、癌基因的活化、氧化应激等）中，还是会被细胞老化诱导而不可逆地停止增殖。

因此，我们认为细胞老化与细胞凋亡相同，有防止异常细胞增殖、抑制癌症的作用。

然而，细胞老化不同于细胞死亡，老化细胞会在生物体内长期生存，因此随着年龄的增长，体内的老化细胞会越来越多。

另一方面，老化细胞中的染色质结构会根据持续的DNA损伤应答而改变，如炎症性细胞因子、趋化因子、基质分解酶和增殖因子等各种炎症蛋白基因活化表达。

已知老化细胞会分泌炎症蛋白到细胞外，这样的细胞老化的表现型被称为SASP（Senescence-associated secretory phenotype）。

到9月9日，社保基金正式进入股市整整3个月，按照有关规定，社保基金必须通过基金管理公司在三个月内完成建仓，并且其持仓市值要达到投资组合总市值80%的水平。

与此前大受追捧的QFII概念相比，社保基金及其所持有的股票显然低调得多，但是在西南证券分析师田磊看来，至少就目前来看，社保基金无论是在资金规模，还是在持股数量上明显都强于境外投资者，其投资理念和行为更可能给市场带来影响。

基金操作的社保基金的选股思路并不侧重某个行业，而更看重企业本身的发展和成长性，并且现阶段的企业经营业绩和走势也不是基金重点考虑的方面。

目前入市的社保基金都是委托南方、博时、华夏、鹏华、长盛、嘉实6家基金管理公司管理。

社保基金大致是被分为14个组合由以上6家管理公司分别管理，每个组合都有一个三位数的代码，第一位代表投资方向，其中“1”指股票投资、“2”指债券投资；第三位数字则代表基金公司名称，其中“1”为南方、“2”为博时、“3”为华夏、“4”为鹏华、“5”为长盛、“6”为嘉实；另有107、108组合主要运作社保基金此前一直持有的中石化股票，分别由博时与华夏基金公司管理。

在许多社保基金介入的股票中经常可以看到开放式基金的身影，例如在被社保基金大量持有的安阳钢铁(600569)的前10大股东中，其第2、6、7、8、9大股东均为开放式基金，而社保基金则以持股500多万股位列第3大股东。

类似的情况也出现在社保基金103组合所持有的华菱管线(000932)上，其第二大股东即为鹏华行业成长证券投资基金，社保基金则以200多万股的持仓量位列第7大股东，此外，在其前10大股东中还有5家是封闭式基金。

对此，某基金公司人士解释说，在获得社保基金管理人资格后，6家基金公司成立了专门的机构理财部门负责社保基金的投资管理，但是其研究、交易系统等则与公募基金共用一个平台，因此社保基金和开放式基金在选股时才会如此一致。

针对“社保概念股”的走势，国盛证券的分析师王剑认为，虽然社保基金此次委托入市资金超过百亿元，但大部分投向是债券，而且由于社保基金的特殊地位，因此基金管理公司对社保基金的操纵策略应该是以“集中持股，稳定股价”为主，不大可能博取太高的收益。

小学上册英语第一单元综合卷英语试题一、综合题(本题有100小题，每小题1分，共100分.每小题不选、错误，均不给分)1.I like to watch ________ in the summer.2.My favorite holiday is ________ (圣诞节). I like to decorate the ________ (圣诞树).3.My favorite book is ________.4.What do we call the place where you can buy groceries?A. StoreB. MarketC. MallD. Supermarket5.The _______ of a balloon can be affected by altitude.6.The _______ (兔子) hops around quickly when it is excited.7.What is the name of the game where you shoot hoops?A. SoccerB. BasketballC. BaseballD. TennisB8. A thermochemical reaction involves heat and chemical ______.9. (85) is a famous park in New York City. The ____10.The _______ (Apollo 11) mission successfully landed humans on the Moon.11.What is 100 - 25?A. 65B. 70C. 75D. 8012.What is the main ingredient in sushi?A. RiceB. NoodlesC. BreadD. PotatoesA13.The bear roams in the _____ woods.14.__________ are important for environmental sustainability.15.The chemical formula for table salt is ______.16.What is the capital of Honduras?A. TegucigalpaB. San Pedro SulaC. La CeibaD. CholutecaA17. A ______ (狗) has a keen sense of smell.18.The ancient Greeks created _______ to explain natural phenomena. (神话)19.The teacher, ______ (老师), guides us in our studies.20.The cake is _______ (刚出炉).21.The _____ (first) man-made satellite was Sputnik, launched by the USSR.22.The capital of Faroe Islands is __________.23.The __________ can provide critical insights into environmental health and stability.24.What do you call the place where we see many books?A. SchoolB. LibraryC. StoreD. Park25.What do you call the study of the Earth's atmosphere?A. MeteorologyB. GeologyC. AstronomyD. Ecology26.What is the term for the distance around a circle?A. AreaB. DiameterC. CircumferenceD. RadiusC27. A ___ (小蝴蝶) flutters gently in the air.28.My ________ (玩具) is made of plush material.29.What do we call the act of cleaning a room?A. TidyingB. OrganizingC. DeclutteringD. CleaningA30.What do we call the tool we use to write on paper?A. MarkerB. PenC. PencilD. All of the above31.The teacher gives _____ (作业) every week.32.The _______ of matter refers to whether it is a solid, liquid, or gas.33.What is the opposite of short?A. TallB. WideC. NarrowD. ThickA34.I like to play ___ (video games).35.I like to play ________ with my friends after school.36.My _____ (表妹) is visiting this weekend.37.The ________ was a famous treaty that settled disputes in Europe.38.What do you call the action of planting flowers in a garden?A. GardeningB. LandscapingC. CultivatingD. SowingA39.ts can live for ______ (数十年). Some pla40.My family lives near a __________ (水库).41.What is the opposite of right?A. WrongB. CorrectC. TrueD. AccurateA42.The _____ (羊) eats grass in the field.43.What is the term for a person who collects stamps?A. PhilatelistB. NumismatistC. CollectorD. HobbyistA44.Every year, we celebrate ______ (感恩节) with a big feast and share what we are thankful for.45.The ancient Egyptians created vast ________ (陵墓) for their pharaohs.46.I have a _____ (遥控车) that can go super fast. 我有一辆可以跑得非常快的遥控车。

Luminescence of (Li 0.333Na 0.334K 0.333)Eu(MoO 4)2and its application in near UV InGaN-based light-emitting diodeZhengliang Wang,Hongbin Liang *,Liya Zhou,Hao Wu,Menglian Gong *,Qiang SuState Key Laboratory of Optoelectronic Materials and Technologies,School of Chemistry and Chemical Engineering,Sun Yat-sen University,Guangzhou,Guangdong 510275,PR ChinaReceived 5May 2005;in final form 1July 2005Available online 26July 2005AbstractA novel red phosphor,(Li 0.333Na 0.334K 0.333)Eu(MoO 4)2(LNKEM),was prepared by solid state reaction technique at high tem-perature.Its photo-luminescent property was investigated and compared with that of Y 2O 2S:0.05Eu 3+,the phosphor currently used in near UV LED (light-emitting diode).It is found that LNKEM shows higher luminescent intensity,and the CIE (Commission Internationale de l ÕEclairage,International Commission on Illumination)chromaticity coordinates of LNKEM is closer to the NTSC standard values than that of Y 2O 2S:0.05Eu 3+.An intense red-emitting LED was fabricated by combining mono-phosphor LNKEM with a $400nm emitting InGaN chip.Ó2005Elsevier B.V.All rights reserved.1.IntroductionMore and more interest was focused on semiconduc-tor-based light-emitting diodes (LEDs)[1–5]since Nakamura and his co-workers [6]fabricated a blue-emitting GaN LED in 1993.Presently,the emission bands of LED chips are shifted to near UV range around 400nm and this wavelength can offer a higher efficiency solid-state lighting [7].The white LED can be generated by several different methods [1–4].The most commonly used method is to combine the red/green/blue tricolor phosphors with a GaN/InGaN chip.The presently used red phosphor for near UV InGaN-based LEDs is mainly Y 2O 2S:Eu 3+[8].But the efficiency of Y 2O 2S:Eu 3+is much lower,compared with the efficiency of the green phosphors (such as ZnS:Cu +/Al 3+)and the blue phosphors (such as BaMgAl 10O 17:Eu 2+)[8].In addition,the lifetime of Y 2O 2S:Eu 3+isinadequate under UV irradiation and it is unstable with releasing of sulfide gas [9].Therefore,it is urgent to search for new red phosphors that can be excited effi-ciently under the near UV range around 400nm with intense emission and appropriate CIE chromaticity coordinates.The appropriate phosphors for near-UV LED must show strong and broad absorption around 400nm firstly.There are two approaches to broaden the absorp-tion in this range.As argued in our previous work [10],one method is by co-doping Sm 3+and Eu 3+ions in the phosphor.It is well known that Sm 3+/Eu 3+ions present strong absorption at about 405/395nm,as a conse-quence,the absorptions around 400nm are expected to be strengthened and broadened by this co-doping sys-tem.On the other hand,from the viewpoint of host compound,each spectroscopic line is expected to be nar-row when the rare earth ions enter the lattice sites of a pure host compound in general.Contrastively,if the host compound can form solid solutions by adjust the cations or anions of this host compound,the sub-lattice structure around the luminescent center ions will be0009-2614/$-see front matter Ó2005Elsevier B.V.All rights reserved.doi:10.1016/j.cplett.2005.07.009*Corresponding authors.Fax:+862084111038.E-mail addresses:cesbin@ (H.Liang),cesgml@ (M.Gong)./locate/cplettChemical Physics Letters 412(2005)313–316expected to be somewhat diverse,and therefore the spec-troscopic lines of rare earth ions are expected to be broadened.In our previous paper[10],we reported the lumines-cence of double molybdates NaLa1Àx Eu x(MoO4)2 and broadened the absorption around400nm by Sm3+–Eu3+co-doped samples,NaSm1Àx Eu x(MoO4)2. These samples show intense red emission,appropriate CIE chromaticity coordinates,and considered to be promising red-emitting component in near UV LED. Because these double molybdates share scheelite-like(CaWO4)iso-structure and no concentration quenching of Eu3+was observed in the series samples of NaLa1Àx Eu x(MoO4)2,as a systematic and further investigations on the near UV LED applications of molybdates red-emitting phosphor,we anticipated to broaden the absorption around400nm by the solidsolution(Li0.333Na0.334K0.333)Eu(MoO4)2(LNKEM). So in present Letter,the luminescence of the sample LNKEM was investigated and compared with that of Y2O2S:Eu3+.Finally,a red LED was fabricated by combining single LNKEM with$400nm InGaN chip.2.ExperimentalThe phosphor(Li0.333Na0.334K0.333)Eu(MoO4)2was prepared by solid state reaction technique at high temper-ature.The stoichiometric mixtures of(NH4)6Mo7O24Æ4-H2O(A.R.grade),Li2CO3(A.R.grade),NaHCO3(A.R. grade),K2CO3(A.R.grade)and Eu2O3(99.99%purity) werefirst ground and pre-fired at500°C for4h,and then heated at800°C for4h.Y2O2S:0.05Eu3+was synthe-sized according to Ref.[11].The structure of thefinal products was examined by X-ray powder diffraction(XRD)using Cu K a radiation on RIGAKU D/max2200vpc X-ray diffractometer. Particle sizes and shapes were observed by scan electron microscopy(SEM)on LEO-1530electron microscope. The excitation and emission spectra of the phosphors were recorded on a JOBIN YVON FL3-21spectrofluo-rometer at room temperature and a450W xenon lamp was used as excitation source.The emission spectrum of the red LED was recorded on Labsphere Inc.LED-1100.3.Results and discussion3.1.XRD and SEM characterizationThe XRD patterns of LNKEM and Y2O2S:0.05Eu3+ were shown in Fig.1.The curve a shows that LNKEM is of single phase and consistent with JCPDS25-0828 [Na0.5Gd0.5MoO4].It reveals that LNKEM shares a tetragonal scheelite structure[12].The XRD pattern of Y2O2S:0.05Eu3+(curve b)is consistent with JCPDS24-1424(Y2O2S)without the presence of Y2O3 phase.It shows that Y2O2S:0.05Eu3+has a hexagonal structure with unit cell dimensions a=0.37nm and c=0.65nm[13].The SEM micrograph of LNKEM was shown in Fig.2.These particles reveal highly crystalline with a diameter of about2l m,which is veryfit to fabricate the solid-lighting devices[14].3.2.Photo-luminescent properties of LNKEM andY2O2S:0.05Eu3+The excitation spectra of Y2O2S:0.05Eu3+(a), LNKEM(b)and NaEu(MoO4)2(c)were shown in Fig.3.Since the purpose of present investigation is on the near UV LED phosphor,only the spectroscopic properties in the range of300–500nm were exhibited in Fig.3.The band from300–390nm in curve a is the Eu3+S2Àcharge transfer(CT)transition in Y2O2S. In curves b and c,the lines in360–500nm range areFig.2.The SEM micrograph of LNKEM.314Z.Wang et al./Chemical Physics Letters412(2005)313–316intra-configurational4f–4f transitions of Eu3+in the host lattices,and the7F0!5L6and7F0!5D2transi-tions at$395and$465nm are two of the strongest absorptions.In last paper[10],we reported the excita-tion spectrum of NaEu(MoO4)2,Comparing the curve b(LNKEM)with curve c[NaEu(MoO4)2],it is obvious that the f–f transitions in curve b was broadened.This may be due to the replacement of partial Na+ions by Li+and K+ions.Alkaline Li+(1s2),Na+(2s22p6)and K+(3s23p6)ions are with similar electronic configura-tions of the noble gases.The ionic radii increase accord-ing to the relative order Li+<Na+<K+,Na+ionic radius is lie between that of Li+and K+.As a result, by partial substitution Na+ions with smaller Li+and bigger K+in appropriate concentrations,it is probably to obtain solid solutions(Li x Na1ÀxÀy K y)Eu(MoO4)2. Actually,the XRD patterns of LNKEM in Fig.1,which are in good line with that of NaEu(MoO4)2,directly show that without any detectable impurity phase in the sample and confirm LNKEM is a solid solution. On the other hand,we prepared the pure single com-pounds MEu(MoO4)2(M=Li,Na,K)also,and indeed find that both LiEu(MoO4)2and NaEu(MoO4)2share tetragonal scheelite[12],whereas KEu(MoO4)2has an order scheelite structure which is triclinic system [15,16].For MEu(MoO4)2(M=Li,Na),the alkali metal ions and rare earth ions are disordered in the same site.Mo(VI)is coordinated by four oxygen atoms in a tet-rahedral site and the alkali metal ion/rare earth ion site is eight coordinated with two sets of rare-oxygen dis-tances[8].In KEu(MoO4)2,Eu3+polyhedra are ordered in sheets,forming a two-dimensional sub-lattice,and its site symmetry is C1[15].In general consideration,if the compound contain below about5%of some secondary phase,it would be non-detectable on the X-ray patterns. Because MEu(MoO4)2(M=Li,Na)and KEu(MoO4)2are of different structure,provided that KEu(MoO4)2 exists as a secondary phase in the sample,it would be about30%level in the sample.This higher secondary phase is easy to be found in XRD patterns.Hence,we believe LNKEM is a solid solution with tetragonal scheelite structure and without impurity phase.M+ (M=Li,Na,K)is next-near coordination cations of Eu3+ions in the sample,they are with similar electronic configurations,same electronic charge Z and different ionic radii r,and therefore they show different ionic po-tential Z/r.The different of ionic radii r or ionic poten-tial Z/r will result in the sub-lattice structure around the luminescent center ions show somewhat diverse.The variation of sub-lattice structure will slight influence the spin–orbit coupling and crystalfield on Eu3+ions, and therefore the spectroscopic lines of Eu3+ions are broadened comparing the single pure compounds.The emission spectra of LNKEM and Y2O2S: 0.05Eu3+under395nm light excitation were shown in Fig.4.The main emission line in curve a is5D0!7F2 transition of Eu3+at616nm,other transitions from the5D J excited levels to7F J ground states,such as 5D!7F J lines in570–720nm range and5D1!7F J transitions in520–570nm range are very weak,which is advantageous to obtain the good CIE chromaticity coordinates.The results imply that Eu3+ions occupy the lattice sites without inversion symmetry,which is in good agreement with the structural results[12]. Y2O2S:0.05Eu3+has a hexagonal structure and the point symmetry of the yttrium site is C3V(3m).Eu3+ was expected to occupy Y3+site in Y2O2S:0.05Eu3+. The main emission peaks at627and616nm of Y2O2S:0.05Eu3+in curve b are ascribed to Eu3+transi-tion from5D0to7F2and its strongest peak is at627nm, other transitions from the5D J(J=0,1,2,3)excited levels to7F J(J=0,1,2,3,4,5,6)ground states are very weak. Comparing Fig.4a with Fig.4b,the following results can be found.First,the emission intensity of LNKEMZ.Wang et al./Chemical Physics Letters412(2005)313–316315under395nm irradiation is about5.4times higher than that of Y2O2S:0.05Eu3+.Second,the CIE chromaticity coordinates are calculated to be x=0.65,y=0.35for LNKEM and x=0.63,y=0.35for Y2O2S:0.05Eu3+. Compared with the NTSC standard CIE chromaticity coordinate values for red(x=0.67,y=0.33),it was found that the CIE chromaticity coordinates of LNKEM was closer to the NTSC standard values than that of Y2O2S:0.05Eu3+.These results imply that the luminescent properties of LNKEM may be better than that of Y2O2S:0.05Eu3+when they are applied in LED.3.3.Fabricate LED with LNKEMOur purpose is to obtain a highly efficient red compo-nent for LED,so a red light-emitting LED was fabri-cated with LNKEM as phosphor.Fig.5shows the emission spectrum of the red light-emitting diode of near UV InGaN-based LNKEM.The band at$395nm is attributed to the emission of InGaN chip and the sharp peaks at616and702nm are due to the emissions of LNKEM.Bright red light from the LED is observed by naked eyes.Its CIE chromaticity coordinates are cal-culated to be x=0.56,y=0.27.The intensive$400nm emission of InGaN chip can still be observed in Fig.5.It is advantageous to obtain a white-emitting LED by combining this phosphor with appropriate blue and green phosphors since the most commonly used method is to combine red/green/blue tricolor phosphors with a GaN/InGaN chip[2].From the standpoint of application,each proper mono-color LED phosphor must meet the following necessary con-ditions.(1)The phosphor must efficiently absorb the 400nm excitation energy that InGaN chip emitted. But any mono-color phosphor cannot absorb all this en-ergy;otherwise,other phosphor probably cannot be effi-ciently excited.(2)The phosphor exhibits higher luminescent intensity under$400nm excitation.(3)The chromaticity coordinates of the phosphor are close to the NTSC standard values.Since LNKEM meets all these conditions,it is con-sidered to be a good candidate for the red component of a three-band white LED.4.ConclusionsDouble molybdates,(Li0.333Na0.334K0.333)Eu(MoO4)2 (LNKEM),is an excellent red-emitting LED phosphor due to its suitable particle size,broadened excitation band in near UV range,intense red-emission with its appropriate CIE chromaticity coordinates.An intense red-emitting LED was successfully fabricated by com-bining single LNKEM with a$400nm emitting InGaN chip.AcknowledgmentThis work wasfinancially supported by a research grant from the Guangdong province government (ZB2003A07).References[1]R.Mueller-Mach,G.O.Mueller,M.R.Krames,T.Trottier,IEEEJ.Select.Top.Quant.Elect.8(2002)339.[2]J.K.Sheu,S.J.Chang,C.H.Kuo,Y.K.Su,L.W.Wu,Y.C.Lin,i,J.M.Tsai,G.C.Chi,R.K.Wu,IEEE Photon.Technol.Lett.15(2003)18.[3]J.K.Park,C.H.Kim,S.H.Park,H.D.Park,S.Y.Choi,Appl.Phys.Lett.84(2004)1647.[4]S.Dalmasso,B.Damilano,C.Pernot,A.Dussaigne,D.Byrne,N.Grandjean,M.Leroux,J.Massies,Phys.Stat.Sol.(a)192(2002) 139.[5]J.Kovac,L.Peternai,O.Lengyel,Thin Solid Films433(2003)22.[6]S.Nakamura,M.Senoh,T.Mukai,Appl.Phys.Lett.62(1993)2390.[7]D.A.Steigerwald,J.C.Bhat,D.Collins,R.M.Fletcher,M.O.Holcomb,M.J.Ludowise,P.S.Martin,S.L.Rudaz,IEEE J.Select.Top.Quant.Elect.8(2002)310.[8]S.Neeraj,N.Kijima,A.K.Cheetham,Chem.Phys.Lett.387(2004)2.[9]T.R.N.Kutty,A.Nag,J.Mater.Chem.13(2003)2271.[10]Z.Wang,H.Liang,M.Gong,Q.Su,Electrochem.Solid-StateLett.8(2005)H33.[11]K.R.Reddy,K.Annapurna,S.Buddhudu,Mater.Res.Bull.31(1996)1355.[12]Y.K.VoronÕko, E.V.Zharikov, D.A.Lis, A.A.Sobol,K.A.Subbotin,hakov,V.E.Shukshin,Proc.SPIE5478(2004)60.[13]Y.-H.Tseng,B.-S.Chiou,C.-C.Peng,L.Ozawa,Thin Solid Films330(1998)173.[14]R.P.Rao,J.Electrochem.Soc.143(1996)189.[15]J.P.M.Van Vliet,G.Blasse,L.H.Brixner,J.Solid State Chem.76(1988)160.[16]H.Yamamoto,S.Seki,T.Ishiba,J.Solid State Chem.94(1991)396.316Z.Wang et al./Chemical Physics Letters412(2005)313–316。

动物科学现代农业科技2011年第21期大口黑鲈(Micropterus salmoides ),俗称加州鲈,原产于美国加利福尼亚州,隶属鲈形目(Perciformes ),太阳鱼科(Ceutrarchidae )。

20世纪80年代初引入我国,由于其生长快、病害少、耐低温、肉多刺少、味道鲜美及营养丰富等优点,已成为我国养殖的主要淡水鱼品种之一。

大口黑鲈属典型淡水肉食性鱼,迄今尚未成功开发出营养平衡的全价专用饲料,尤其全程使用饲料一直是业界的一大难题,表现在中后期经常出现生长慢、厌食、肝脏疾病等问题[1]。

虽然大口黑鲈的养殖在国内外均有一定的规模,而且饲料成本占养殖成本的比例较高,但有关大口黑鲈营养需要的研究仍十分缺乏[2]。

在国外,大部分大口黑鲈的养殖,均采用比较容易获得的其他肉食性鱼类如鲑鱼和鳟鱼的饲料,而非采用针对大口黑鲈自身营养需要配制的专用饲料[3]。

在国内,养殖户投喂的饲料多以冰鲜下杂鱼和其他动物性饲料为主,这对海洋资源无疑是一种浪费,同时对养殖环境的污染也十分明显,容易引起各种疾病的暴发[4]。

按大口黑鲈2010年的产量测算,我国潜在的鲈鱼专用饲料需求可达20万t/年[1]。

对配合饲料的需要日益增加,亟待进一步全面开展其营养需要的研究。

因此,该文综述了国外内大口黑鲈营养需要的研究进展,并参考其他鱼类的营养需要,比较全面地总结了大口黑鲈对饲料中各营养素的需要量,以期为大口黑鲈专用饲料的研发和配制提供参考。

1大黑鲈对各种营养成分的需要量1.1蛋白质和氨基酸由于没有专门为大口黑鲈开发的商用饲料,目前在国外均采用其他肉性鱼类的饲料(蛋白质含量>40%,鱼粉含量50%~70%)[5-8]。

最早关于大口黑鲈饲料蛋白质营养需要的研究见于1981年[5]。

研究发现,0~1龄的大口黑鲈对饲料中蛋白质的需要量为39.9%~40.8%(基于饲料干物质)。

以饲料中水分含量为10%来计算的话,蛋白质含量为36%~37%(饲料湿重)即可满足1龄及之前的大口黑鲈鱼的生长。

血清残余胆固醇水平对冠心病的影响及临床意义陈翠1，2，杨莉婷3，唐陶1，2，徐浩2，刘茂41.川北医学院附属医院遗传与产前诊断中心，四川南充637000；2.川北医学院检验医学院，四川南充637000；3.南充市中心医院检验科，四川南充637000；4.川北医学院附属医院心血管内科，四川南充637000【摘要】目的探讨血清残余胆固醇(RC)水平对冠心病的影响及其临床意义。

方法回顾性分析2019年6月至2020年6月因胸闷胸痛于川北医学院附属医院心内科住院行冠脉造影检查的230例患者的临床资料，根据冠脉造影结果分为冠心病组190例和非冠心病组40例(CON 组)，根据临床诊断标准又将冠心病患者分为稳定性心绞痛组(SAP 组)70例和急性冠脉综合征组(ACS 组)120例。

比较三组患者的一般资料和RC 水平，采用Spearman 秩相关分析RC 水平与Gensini 评分的相关性，采用多因素Logistic 回归分析影响冠心病发生的风险因素，绘制受试者工作特征曲线(ROC)分析RC 对冠心病发生的预测价值。

结果ACS 组、SAP 组和CON 组患者的性别、年龄、吸烟史、高血压史、LP(a)比较差异均无统计学意义(P >0.05)，但ACS 组和SAP 组患者的总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、载脂蛋白A (ApoA)、载脂蛋白B (ApoB)水平明显高于CON 组，而高密度脂蛋白胆固醇(HDL-C)水平明显低于CON 组，且ACS 组患者的TC 、LDL-C 、ApoB 水平明显高于SAP 组，差异均有统计学意义(P <0.05)；ACS 组和SAP 组患者的RC 水平分别为(0.98±0.37)mmol/L 、(0.86±0.23)mmol/L ，明显高于CON 组的(0.68±0.16)mmol/L ，且ACS 组的RC 水平明显高于SAP 组，差异均具有统计学意义(P <0.05)；经Spearman 秩相关分析结果显示，RC 水平与Gensini 评分呈正相关(P <0.05)；经多因素Logistic 回归分析结果显示，年龄、吸烟、RC 、LDL-C 、ApoA 为冠心病的独立危险因素(P <0.05)；经ROC 分析结果显示，血清RC 预测冠心病发生的曲线下面积(AUC)为0.755，灵敏性和特异性分别为53.20%和87.50%。

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic e aerobic sequencing batch reactorZhang Bin a ,b ,Chen Zhe a ,b ,Qiu Zhigang a ,b ,Jin Min a ,b ,Chen Zhiqiang a ,b ,Chen Zhaoli a ,b ,Li Junwen a ,b ,Wang Xuan c ,*,Wang Jingfeng a ,b ,**aInstitute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China bTianjin Key Laboratory of Risk Assessment and Control for Environment and Food Safety,Tianjin 300050,PR China cTianjin Key Laboratory of Hollow Fiber Membrane Material and Membrane Process,Institute of Biological and Chemical Engineering,Tianjin Polytechnical University,Tianjin 300160,PR Chinaa r t i c l e i n f oArticle history:Received 30June 2011Received in revised form 10September 2011Accepted 10September 2011Available online xxx Keywords:Ammonia-oxidizing bacteria Granular sludgeCommunity development Granule sizeNitrifying bacteria distribution Phylogenetic diversitya b s t r a c tThe structure dynamic of ammonia-oxidizing bacteria (AOB)community and the distribution of AOB and nitrite-oxidizing bacteria (NOB)in granular sludge from an anaerobic e aerobic sequencing batch reactor (SBR)were investigated.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and characterize these organisms.The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge.Along with granules formation,the AOB diversity declined due to the selection pressure imposed by process conditions.Denaturing gradient gel electrophoresis (DGGE)and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling e washing out action.Furthermore,DGGE analysis revealed that larger granules benefit more AOB species surviving in the reactor.In the SBR were various size granules coexisted,granule diameter affected the distribution range of AOB and NOB.Small and medium granules (d <0.6mm)cannot restrict oxygen mass transfer in all spaces of the rger granules (d >0.9mm)can result in smaller aerobic volume fraction and inhibition of NOB growth.All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.ª2011Elsevier Ltd.All rights reserved.1.IntroductionAt sufficiently high levels,ammonia in aquatic environments can be toxic to aquatic life and can contribute to eutrophica-tion.Accordingly,biodegradation and elimination of ammonia in wastewater are the primary functions of thewastewater treatment process.Nitrification,the conversion of ammonia to nitrate via nitrite,is an important way to remove ammonia nitrogen.It is a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and NOB).Aerobic ammonia-oxidation is often the first,rate-limiting step of nitrification;however,it is essential for the*Corresponding author .**Corresponding author.Institute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China.Tel.:+862284655498;fax:+862223328809.E-mail addresses:wangxuan0116@ (W.Xuan),jingfengwang@ (W.Jingfeng).Available online atjournal homepage:/locate/watresw a t e r r e s e a r c h x x x (2011)1e 100043-1354/$e see front matter ª2011Elsevier Ltd.All rights reserved.doi:10.1016/j.watres.2011.09.026removal of ammonia from the wastewater(Prosser and Nicol, 2008).Comparative analyses of16S rRNA sequences have revealed that most AOB in activated sludge are phylogeneti-cally closely related to the clade of b-Proteobacteria (Kowalchuk and Stephen,2001).However,a number of studies have suggested that there are physiological and ecological differences between different AOB genera and lineages,and that environmental factors such as process parameter,dis-solved oxygen,salinity,pH,and concentrations of free ammonia can impact certain species of AOB(Erguder et al., 2008;Kim et al.,2006;Koops and Pommerening-Ro¨ser,2001; Kowalchuk and Stephen,2001;Shi et al.,2010).Therefore, the physiological activity and abundance of AOB in waste-water processing is critical in the design and operation of waste treatment systems.For this reason,a better under-standing of the ecology and microbiology of AOB in waste-water treatment systems is necessary to enhance treatment performance.Recently,several developed techniques have served as valuable tools for the characterization of microbial diversity in biological wastewater treatment systems(Li et al., 2008;Yin and Xu,2009).Currently,the application of molec-ular biotechniques can provide clarification of the ammonia-oxidizing community in detail(Haseborg et al.,2010;Tawan et al.,2005;Vlaeminck et al.,2010).In recent years,the aerobic granular sludge process has become an attractive alternative to conventional processes for wastewater treatment mainly due to its cell immobilization strategy(de Bruin et al.,2004;Liu et al.,2009;Schwarzenbeck et al.,2005;Schwarzenbeck et al.,2004a,b;Xavier et al.,2007). Granules have a more tightly compact structure(Li et al.,2008; Liu and Tay,2008;Wang et al.,2004)and rapid settling velocity (Kong et al.,2009;Lemaire et al.,2008).Therefore,granular sludge systems have a higher mixed liquid suspended sludge (MLSS)concentration and longer solid retention times(SRT) than conventional activated sludge systems.Longer SRT can provide enough time for the growth of organisms that require a long generation time(e.g.,AOB).Some studies have indicated that nitrifying granules can be cultivated with ammonia-rich inorganic wastewater and the diameter of granules was small (Shi et al.,2010;Tsuneda et al.,2003).Other researchers reported that larger granules have been developed with the synthetic organic wastewater in sequencing batch reactors(SBRs)(Li et al., 2008;Liu and Tay,2008).The diverse populations of microor-ganisms that coexist in granules remove the chemical oxygen demand(COD),nitrogen and phosphate(de Kreuk et al.,2005). However,for larger granules with a particle diameter greater than0.6mm,an outer aerobic shell and an inner anaerobic zone coexist because of restricted oxygen diffusion to the granule core.These properties of granular sludge suggest that the inner environment of granules is unfavorable to AOB growth.Some research has shown that particle size and density induced the different distribution and dominance of AOB,NOB and anam-mox(Winkler et al.,2011b).Although a number of studies have been conducted to assess the ecology and microbiology of AOB in wastewater treatment systems,the information on the dynamics,distribution,and quantification of AOB communities during sludge granulation is still limited up to now.To address these concerns,the main objective of the present work was to investigate the population dynamics of AOB communities during the development of seedingflocs into granules,and the distribution of AOB and NOB in different size granules from an anaerobic e aerobic SBR.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and char-acterize these organisms.Based on these approaches,we demonstrate the differences in both AOB community evolu-tion and composition of theflocs and granules co-existing in the SBR and further elucidate the relationship between distribution of nitrifying bacteria and granule size.It is ex-pected that the work would be useful to better understand the mechanisms responsible for the AOB in granules and apply them for optimal control and management strategies of granulation systems.2.Material and methods2.1.Reactor set-up and operationThe granules were cultivated in a lab-scale SBR with an effective volume of4L.The effective diameter and height of the reactor was10cm and51cm,respectively.The hydraulic retention time was set at8h.Activated sludge from a full-scale sewage treat-ment plant(Jizhuangzi Sewage Treatment Works,Tianjin, China)was used as the seed sludge for the reactor at an initial sludge concentration of3876mg LÀ1in MLSS.The reactor was operated on6-h cycles,consisting of2-min influent feeding,90-min anaerobic phase(mixing),240-min aeration phase and5-min effluent discharge periods.The sludge settling time was reduced gradually from10to5min after80SBR cycles in20days, and only particles with a settling velocity higher than4.5m hÀ1 were retained in the reactor.The composition of the influent media were NaAc(450mg LÀ1),NH4Cl(100mg LÀ1),(NH4)2SO4 (10mg LÀ1),KH2PO4(20mg LÀ1),MgSO4$7H2O(50mg LÀ1),KCl (20mg LÀ1),CaCl2(20mg LÀ1),FeSO4$7H2O(1mg LÀ1),pH7.0e7.5, and0.1mL LÀ1trace element solution(Li et al.,2007).Analytical methods-The total organic carbon(TOC),NHþ4e N, NOÀ2e N,NOÀ3e N,total nitrogen(TN),total phosphate(TP) concentration,mixed liquid suspended solids(MLSS) concentration,and sludge volume index at10min(SVI10)were measured regularly according to the standard methods (APHA-AWWA-WEF,2005).Sludge size distribution was determined by the sieving method(Laguna et al.,1999).Screening was performed with four stainless steel sieves of5cm diameter having respective mesh openings of0.9,0.6,0.45,and0.2mm.A100mL volume of sludge from the reactor was sampled with a calibrated cylinder and then deposited on the0.9mm mesh sieve.The sample was subsequently washed with distilled water and particles less than0.9mm in diameter passed through this sieve to the sieves with smaller openings.The washing procedure was repeated several times to separate the gran-ules.The granules collected on the different screens were recovered by backwashing with distilled water.Each fraction was collected in a different beaker andfiltered on quantitative filter paper to determine the total suspended solid(TSS).Once the amount of total suspended solid(TSS)retained on each sieve was acquired,it was reasonable to determine for each class of size(<0.2,[0.2e0.45],[0.45e0.6],[0.6e0.9],>0.9mm) the percentage of the total weight that they represent.w a t e r r e s e a r c h x x x(2011)1e10 22.2.DNA extraction and nested PCR e DGGEThe sludge from approximately8mg of MLSS was transferred into a1.5-mL Eppendorf tube and then centrifuged at14,000g for10min.The supernatant was removed,and the pellet was added to1mL of sodium phosphate buffer solution and aseptically mixed with a sterilized pestle in order to detach granules.Genomic DNA was extracted from the pellets using E.Z.N.A.äSoil DNA kit(D5625-01,Omega Bio-tek Inc.,USA).To amplify ammonia-oxidizer specific16S rRNA for dena-turing gradient gel electrophoresis(DGGE),a nested PCR approach was performed as described previously(Zhang et al., 2010).30m l of nested PCR amplicons(with5m l6Âloading buffer)were loaded and separated by DGGE on polyacrylamide gels(8%,37.5:1acrylamide e bisacrylamide)with a linear gradient of35%e55%denaturant(100%denaturant¼7M urea plus40%formamide).The gel was run for6.5h at140V in 1ÂTAE buffer(40mM Tris-acetate,20mM sodium acetate, 1mM Na2EDTA,pH7.4)maintained at60 C(DCodeäUniversal Mutation Detection System,Bio-Rad,Hercules,CA, USA).After electrophoresis,silver-staining and development of the gels were performed as described by Sanguinetti et al. (1994).These were followed by air-drying and scanning with a gel imaging analysis system(Image Quant350,GE Inc.,USA). The gel images were analyzed with the software Quantity One,version4.31(Bio-rad).Dice index(Cs)of pair wise community similarity was calculated to evaluate the similarity of the AOB community among DGGE lanes(LaPara et al.,2002).This index ranges from0%(no common band)to100%(identical band patterns) with the assistance of Quantity One.The Shannon diversity index(H)was used to measure the microbial diversity that takes into account the richness and proportion of each species in a population.H was calculatedusing the following equation:H¼ÀPn iNlogn iN,where n i/Nis the proportion of community made up by species i(bright-ness of the band i/total brightness of all bands in the lane).Dendrograms relating band pattern similarities were automatically calculated without band weighting(consider-ation of band density)by the unweighted pair group method with arithmetic mean(UPGMA)algorithms in the Quantity One software.Prominent DGGE bands were excised and dissolved in30m L Milli-Q water overnight,at4 C.DNA was recovered from the gel by freeze e thawing thrice.Cloning and sequencing of the target DNA fragments were conducted following the estab-lished method(Zhang et al.,2010).2.3.Distribution of nitrifying bacteriaThree classes of size([0.2e0.45],[0.45e0.6],>0.9mm)were chosen on day180for FISH analysis in order to investigate the spatial distribution characteristics of AOB and NOB in granules.2mg sludge samples werefixed in4%para-formaldehyde solution for16e24h at4 C and then washed twice with sodium phosphate buffer;the samples were dehydrated in50%,80%and100%ethanol for10min each. Ethanol in the granules was then completely replaced by xylene by serial immersion in ethanol-xylene solutions of3:1, 1:1,and1:3by volume andfinally in100%xylene,for10min periods at room temperature.Subsequently,the granules were embedded in paraffin(m.p.56e58 C)by serial immer-sion in1:1xylene-paraffin for30min at60 C,followed by 100%paraffin.After solidification in paraffin,8-m m-thick sections were prepared and placed on gelatin-coated micro-scopic slides.Paraffin was removed by immersing the slide in xylene and ethanol for30min each,followed by air-drying of the slides.The three oligonucleotide probes were used for hybridiza-tion(Downing and Nerenberg,2008):FITC-labeled Nso190, which targets the majority of AOB;TRITC-labeled NIT3,which targets Nitrobacter sp.;TRITC-labeled NSR1156,which targets Nitrospira sp.All probe sequences,their hybridization condi-tions,and washing conditions are given in Table1.Oligonu-cleotides were synthesized andfluorescently labeled with fluorochomes by Takara,Inc.(Dalian,China).Hybridizations were performed at46 C for2h with a hybridization buffer(0.9M NaCl,formamide at the percentage shown in Table1,20mM Tris/HCl,pH8.0,0.01% SDS)containing each labeled probe(5ng m LÀ1).After hybrid-ization,unbound oligonucleotides were removed by a strin-gent washing step at48 C for15min in washing buffer containing the same components as the hybridization buffer except for the probes.For detection of all DNA,4,6-diamidino-2-phenylindole (DAPI)was diluted with methanol to afinal concentration of1ng m LÀ1.Cover the slides with DAPI e methanol and incubate for15min at37 C.The slides were subsequently washed once with methanol,rinsed briefly with ddH2O and immediately air-dried.Vectashield(Vector Laboratories)was used to prevent photo bleaching.The hybridization images were captured using a confocal laser scanning microscope (CLSM,Zeiss710).A total of10images were captured for each probe at each class of size.The representative images were selected andfinal image evaluation was done in Adobe PhotoShop.w a t e r r e s e a r c h x x x(2011)1e1033.Results3.1.SBR performance and granule characteristicsDuring the startup period,the reactor removed TOC and NH 4þ-N efficiently.98%of NH 4þ-N and 100%of TOC were removed from the influent by day 3and day 5respectively (Figs.S2,S3,Supporting information ).Removal of TN and TP were lower during this period (Figs.S3,S4,Supporting information ),though the removal of TP gradually improved to 100%removal by day 33(Fig.S4,Supporting information ).To determine the sludge volume index of granular sludge,a settling time of 10min was chosen instead of 30min,because granular sludge has a similar SVI after 60min and after 5min of settling (Schwarzenbeck et al.,2004b ).The SVI 10of the inoculating sludge was 108.2mL g À1.The changing patterns of MLSS and SVI 10in the continuous operation of the SBR are illustrated in Fig.1.The sludge settleability increased markedly during the set-up period.Fig.2reflects the slow andgradual process of sludge granulation,i.e.,from flocculentsludge to granules.3.2.DGGE analysis:AOB communities structure changes during sludge granulationThe results of nested PCR were shown in Fig.S1.The well-resolved DGGE bands were obtained at the representative points throughout the GSBR operation and the patterns revealed that the structure of the AOB communities was dynamic during sludge granulation and stabilization (Fig.3).The community structure at the end of experiment was different from that of the initial pattern of the seed sludge.The AOB communities on day 1showed 40%similarity only to that at the end of the GSBR operation (Table S1,Supporting information ),indicating the considerable difference of AOB communities structures between inoculated sludge and granular sludge.Biodiversity based on the DGGE patterns was analyzed by calculating the Shannon diversity index H as204060801001201401254159738494104115125135147160172188Time (d)S V I 10 (m L .g -1)10002000300040005000600070008000900010000M L S S (m g .L -1)Fig.1e Change in biomass content and SVI 10during whole operation.SVI,sludge volume index;MLSS,mixed liquid suspendedsolids.Fig.2e Variation in granule size distribution in the sludge during operation.d,particle diameter;TSS,total suspended solids.w a t e r r e s e a r c h x x x (2011)1e 104shown in Fig.S5.In the phase of sludge inoculation (before day 38),H decreased remarkably (from 0.94to 0.75)due to the absence of some species in the reactor.Though several dominant species (bands2,7,10,11)in the inoculating sludge were preserved,many bands disappeared or weakened (bands 3,4,6,8,13,14,15).After day 45,the diversity index tended to be stable and showed small fluctuation (from 0.72to 0.82).Banding pattern similarity was analyzed by applying UPGMA (Fig.4)algorithms.The UPGMA analysis showed three groups with intragroup similarity at approximately 67%e 78%and intergroup similarity at 44e 62%.Generally,the clustering followed the time course;and the algorithms showed a closer clustering of groups II and III.In the analysis,group I was associated with sludge inoculation and washout,group IIwithFig.3e DGGE profile of the AOB communities in the SBR during the sludge granulation process (lane labels along the top show the sampling time (days)from startup of the bioreactor).The major bands were labeled with the numbers (bands 1e15).Fig.4e UPGMA analysis dendrograms of AOB community DGGE banding patterns,showing schematics of banding patterns.Roman numerals indicate major clusters.w a t e r r e s e a r c h x x x (2011)1e 105startup sludge granulation and decreasing SVI 10,and group III with a stable system and excellent biomass settleability.In Fig.3,the locations of the predominant bands were excised from the gel.DNA in these bands were reamplified,cloned and sequenced.The comparative analysis of these partial 16S rRNA sequences (Table 2and Fig.S6)revealed the phylogenetic affiliation of 13sequences retrieved.The majority of the bacteria in seed sludge grouped with members of Nitrosomonas and Nitrosospira .Along with sludge granula-tion,most of Nitrosomonas (Bands 2,5,7,9,10,11)were remained or eventually became dominant in GSBR;however,all of Nitrosospira (Bands 6,13,15)were gradually eliminated from the reactor.3.3.Distribution of AOB and NOB in different sized granulesFISH was performed on the granule sections mainly to deter-mine the location of AOB and NOB within the different size classes of granules,and the images were not further analyzed for quantification of cell counts.As shown in Fig.6,in small granules (0.2mm <d <0.45mm),AOB located mainly in the outer part of granular space,whereas NOB were detected only in the core of granules.In medium granules (0.45mm <d <0.6mm),AOB distributed evenly throughout the whole granular space,whereas NOB still existed in the inner part.In the larger granules (d >0.9mm),AOB and NOB were mostly located in the surface area of the granules,and moreover,NOB became rare.4.Discussion4.1.Relationship between granule formation and reactor performanceAfter day 32,the SVI 10stabilized at 20e 35mL g À1,which is very low compared to the values measured for activated sludge (100e 150mL g À1).However,the size distribution of the granules measured on day 32(Fig.2)indicated that only 22%of the biomass was made of granular sludge with diameter largerthan 0.2mm.These results suggest that sludge settleability increased prior to granule formation and was not affected by different particle sizes in the sludge during the GSBR operation.It was observed,however,that the diameter of the granules fluctuated over longer durations.The large granules tended to destabilize due to endogenous respiration,and broke into smaller granules that could seed the formation of large granules again.Pochana and Keller reported that physically broken sludge flocs contribute to lower denitrification rates,due to their reduced anoxic zone (Pochana and Keller,1999).Therefore,TN removal efficiency raises fluctuantly throughout the experiment.Some previous research had demonstrated that bigger,more dense granules favored the enrichment of PAO (Winkler et al.,2011a ).Hence,after day 77,removal efficiency of TP was higher and relatively stable because the granules mass fraction was over 90%and more larger granules formed.4.2.Relationship between AOB communities dynamic and sludge granulationFor granule formation,a short settling time was set,and only particles with a settling velocity higher than 4.5m h À1were retained in the reactor.Moreover,as shown in Fig.1,the variation in SVI 10was greater before day 41(from 108.2mL g À1e 34.1mL g À1).During this phase,large amounts of biomass could not survive in the reactor.A clear shift in pop-ulations was evident,with 58%similarity between days 8and 18(Table S1).In the SBR system fed with acetate-based synthetic wastewater,heterotrophic bacteria can produce much larger amounts of extracellular polysaccharides than autotrophic bacteria (Tsuneda et al.,2003).Some researchers found that microorganisms in high shear environments adhered by extracellular polymeric substances (EPS)to resist the damage of suspended cells by environmental forces (Trinet et al.,1991).Additionally,it had been proved that the dominant heterotrophic species in the inoculating sludge were preserved throughout the process in our previous research (Zhang et al.,2011).It is well known that AOB are chemoau-totrophic and slow-growing;accordingly,numerous AOBw a t e r r e s e a r c h x x x (2011)1e 106populations that cannot become big and dense enough to settle fast were washed out from the system.As a result,the variation in AOB was remarkable in the period of sludge inoculation,and the diversity index of population decreased rapidly.After day 45,AOB communities’structure became stable due to the improvement of sludge settleability and the retention of more biomass.These results suggest that the short settling time (selection pressure)apparently stressed the biomass,leading to a violent dynamic of AOB communities.Further,these results suggest that certain populations may have been responsible for the operational success of the GSBR and were able to persist despite the large fluctuations in pop-ulation similarity.This bacterial population instability,coupled with a generally acceptable bioreactor performance,is congruent with the results obtained from a membrane biore-actor (MBR)for graywater treatment (Stamper et al.,2003).Nitrosomonas e like and Nitrosospira e like populations are the dominant AOB populations in wastewater treatment systems (Kowalchuk and Stephen,2001).A few previous studies revealed that the predominant populations in AOB communities are different in various wastewater treatment processes (Tawan et al.,2005;Thomas et al.,2010).Some researchers found that the community was dominated by AOB from the genus Nitrosospira in MBRs (Zhang et al.,2010),whereas Nitrosomonas sp.is the predominant population in biofilter sludge (Yin and Xu,2009).In the currentstudy,Fig.5e DGGE profile of the AOB communities in different size of granules (lane labels along the top show the range of particle diameter (d,mm)).Values along the bottom indicate the Shannon diversity index (H ).Bands labeled with the numbers were consistent with the bands in Fig.3.w a t e r r e s e a r c h x x x (2011)1e 107sequence analysis revealed that selection pressure evidently effect on the survival of Nitrosospira in granular sludge.Almost all of Nitrosospira were washed out initially and had no chance to evolve with the environmental changes.However,some members of Nitrosomonas sp.have been shown to produce more amounts of EPS than Nitrosospira ,especially under limited ammonia conditions (Stehr et al.,1995);and this feature has also been observed for other members of the same lineage.Accordingly,these EPS are helpful to communicate cells with each other and granulate sludge (Adav et al.,2008).Therefore,most of Nitrosomonas could adapt to this challenge (to become big and dense enough to settle fast)and were retained in the reactor.At the end of reactor operation (day 180),granules with different particle size were sieved.The effects of variation in granules size on the composition of the AOBcommunitiesFig.6e Micrographs of FISH performed on three size classes of granule sections.DAPI stain micrographs (A,D,G);AOB appear as green fluorescence (B,E,H),and NOB appear as red fluorescence (C,F,I).Bar [100m m in (A)e (C)and (G)e (I).d,particle diameter.(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)w a t e r r e s e a r c h x x x (2011)1e 108were investigated.As shown in Fig.5,AOB communities structures in different size of granules were varied.Although several predominant bands(bands2,5,11)were present in all samples,only bands3and6appeared in the granules with diameters larger than0.6mm.Additionally,bands7and10 were intense in the granules larger than0.45mm.According to Table2,it can be clearly indicated that Nitrosospira could be retained merely in the granules larger than0.6mm.Therefore, Nitrosospira was not present at a high level in Fig.3due to the lower proportion of larger granules(d>0.6mm)in TSS along with reactor operation.DGGE analysis also revealed that larger granules had a greater microbial diversity than smaller ones. This result also demonstrates that more organisms can survive in larger granules as a result of more space,which can provide the suitable environment for the growth of microbes(Fig.6).4.3.Effect of variance in particle size on the distribution of AOB and NOB in granulesAlthough an influence of granule size has been observed in experiments and simulations for simultaneous N-and P-removal(de Kreuk et al.,2007),the effect of granule size on the distribution of different biomass species need be revealed further with the assistance of visible experimental results, especially in the same granular sludge reactors.Related studies on the diversity of bacterial communities in granular sludge often focus on the distribution of important functional bacteria populations in single-size granules(Matsumoto et al., 2010).In the present study,different size granules were sieved,and the distribution patterns of AOB and NOB were explored.In the nitrification processes considered,AOB and NOB compete for space and oxygen in the granules(Volcke et al.,2010).Since ammonium oxidizers have a higheroxygen affinity(K AOBO2<K NOBO2)and accumulate more rapidly inthe reactor than nitrite oxidizers(Volcke et al.,2010),NOB are located just below the layer of AOB,where still some oxygen is present and allows ready access to the nitrite produced.In smaller granules,the location boundaries of the both biomass species were distinct due to the limited existence space provided by granules for both microorganism’s growth.AOB exist outside of the granules where oxygen and ammonia are present.Medium granules can provide broader space for microbe multiplying;accordingly,AOB spread out in the whole granules.This result also confirms that oxygen could penetrate deep into the granule’s core without restriction when particle diameter is less than0.6mm.Some mathematic model also supposed that NOBs are favored to grow in smaller granules because of the higher fractional aerobic volume (Volcke et al.,2010).As shown in the results of the batch experiments(Zhang et al.,2011),nitrite accumulation temporarily occurred,accompanied by the more large gran-ules(d>0.9mm)forming.This phenomenon can be attrib-uted to the increased ammonium surface load associated with larger granules and smaller aerobic volume fraction,resulting in outcompetes of NOB.It also suggests that the core areas of large granules(d>0.9mm)could provide anoxic environment for the growth of anaerobic denitrificans(such as Tb.deni-trificans or Tb.thioparus in Fig.S7,Supporting information).As shown in Fig.2and Fig.S3,the removal efficiency of total nitrogen increased with formation of larger granules.5.ConclusionsThe variation in AOB communities’structure was remarkable during sludge inoculation,and the diversity index of pop-ulation decreased rapidly.Most of Nitrosomonas in the inocu-lating sludge were retained because of their capability to rapidly adapt to the settling e washing out action.DGGE anal-ysis also revealed that larger granules had greater AOB diversity than that of smaller ones.Oxygen penetration was not restricted in the granules of less than0.6mm particle diameter.However,the larger granules(d>0.9mm)can result in the smaller aerobic volume fraction and inhibition of NOB growth.Henceforth,further studies on controlling and opti-mizing distribution of granule size could be beneficial to the nitrogen removal and expansive application of granular sludge technology.AcknowledgmentsThis work was supported by grants from the National Natural Science Foundation of China(No.51108456,50908227)and the National High Technology Research and Development Program of China(No.2009AA06Z312).Appendix.Supplementary dataSupplementary data associated with this article can be found in online version at doi:10.1016/j.watres.2011.09.026.r e f e r e n c e sAdav,S.S.,Lee, D.J.,Show,K.Y.,2008.Aerobic granular sludge:recent advances.Biotechnology Advances26,411e423.APHA-AWWA-WEF,2005.Standard Methods for the Examination of Water and Wastewater,first ed.American Public Health Association/American Water Works Association/WaterEnvironment Federation,Washington,DC.de Bruin,L.M.,de Kreuk,M.,van der Roest,H.F.,Uijterlinde,C., van Loosdrecht,M.C.M.,2004.Aerobic granular sludgetechnology:an alternative to activated sludge?Water Science and Technology49,1e7.de Kreuk,M.,Heijnen,J.J.,van Loosdrecht,M.C.M.,2005.Simultaneous COD,nitrogen,and phosphate removal byaerobic granular sludge.Biotechnology and Bioengineering90, 761e769.de Kreuk,M.,Picioreanu,C.,Hosseini,M.,Xavier,J.B.,van Loosdrecht,M.C.M.,2007.Kinetic model of a granular sludge SBR:influences on nutrient removal.Biotechnology andBioengineering97,801e815.Downing,L.S.,Nerenberg,R.,2008.Total nitrogen removal ina hybrid,membrane-aerated activated sludge process.WaterResearch42,3697e3708.Erguder,T.H.,Boon,N.,Vlaeminck,S.E.,Verstraete,W.,2008.Partial nitrification achieved by pulse sulfide doses ina sequential batch reactor.Environmental Science andTechnology42,8715e8720.w a t e r r e s e a r c h x x x(2011)1e109。

Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·1.Description (1)2.Product Components and Storage Conditions (4)3.Performing the CellTiter-Glo ®Assay (5)A.Reagent Preparation (5)B.Protocol for the Cell Viability Assay (6)C.Protocol for Generating an ATP Standard Curve (optional) (7)4.Appendix (7)A.Overview of the CellTiter-Glo ®Assay..............................................................7B.Additional Considerations..................................................................................8C.References............................................................................................................11D.Related Products. (12)1.DescriptionThe CellTiter-Glo ®Luminescent Cell Viability Assay (a–e)is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The CellTiter-Glo ®Assay is designed for use with multiwell-plate formats, making it ideal for automated high-throughput screening (HTS) and cell proliferation and cytotoxicity assays. The homogeneous assay procedure (Figure 1) involves adding a single reagent (CellTiter-Glo ®Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium or multiple pipetting steps are not required.The homogeneous “add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present (Figure 2).The amount of ATP is directly proportional to the number of cells present in culture in agreement with previous reports (1). The CellTiter-Glo ®Assay relies on the properties of a proprietary thermostable luciferase (Ultra-Glo™ Recombinant Luciferase), which generates a stable “glow-type” luminescent signal and improves performance across a wide range of assay conditions. The luciferase reaction for this assay is shown in Figure 3. The half-life of the luminescent signal resulting from this reaction is greater than five hours (Figure 4). This extended half-life eliminates the need for reagent injectors and provides flexibility for continuous or batch-mode processing of multiple plates. The unique homogeneous format reduces pipetting errors that may be introduced during the multiple steps required by other ATP-measurement methods.CellTiter-Glo ®Luminescent Cell Viability AssayAll technical literature is available on the Internet at: /protocols/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useofthissystem.E-mail:********************Figure 1. Flow diagram showing preparation and use of CellTiter-Glo ®Reagent.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3170M A 12_0ACellTiter-Glo CellTiter-Glo MixerLuminometer®System Advantages•Homogeneous:“Add-mix-measure” format reduces the number of plate-handling steps to fewer than that required for similar ATP assays.•Fast:Data can be recorded 10 minutes after adding reagent.•Sensitive:Measures cells at numbers below the detection limits of standard colorimetric and fluorometric assays.•Flexible:Can be used with various multiwell formats. Data can be recorded by luminometer or CCD camera or imaging device.•Robust:Luminescent signal is very stable, with a half-life >5 hours,depending on cell type and culture medium used.•Able to Multiplex:Can be used with reporter gene assays or other cell-based assays from Promega (2,3).Figure 3. The luciferase reaction.Mono-oxygenation of luciferin is catalyzed byluciferase in the presence of Mg 2+, ATP and molecular oxygen.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3171M A 12_0A L u m i n e s c e n c e (R L U )Cells per Well10,00060,00020,00030,00040,00050,0000R² = 0.9990.5 × 1061.0 × 1061.5 × 1062.0 × 1062.5 × 1063.0 × 1063.5 × 1064.0 × 106r² = 0.99020,00010,00030,00040,00050,000r² = 0.9900100200300400HO SN S N O S N S N OCOOH +ATP+O 2Ultra-Glo™ Recombinant Luciferase +AMP+PP i +CO 2+LightBeetle Luciferin OxyluciferinMg 2+0Figure 2. Cell number correlates with luminescent output.A direct relationship exists between luminescence measured with the CellTiter-Glo ®Assay and the number of cells in culture over three orders of magnitude. Serial twofold dilutions of HEK293cells were made in a 96-well plate in DMEM with 10% FBS, and assays wereperformed as described in Section 3.B. Luminescence was recorded 10minutes after reagent addition using a GloMax ®-Multi+ Detection System. Values represent the mean ± S.D. of four replicates for each cell number. The luminescent signal from 50HEK293 cells is greater than three times the background signal from serum-supplemented medium without cells. There is a linear relationship (r 2= 0.99)between the luminescent signal and the number of cells from 0to 50,000 cells per well.Figure 4. Extended luminescent half-life allows high-throughput batchprocessing.Signal stability is shown for three common cell lines. HepG2 and BHK-21cells were grown and assayed in MEM containing 10% FBS, while CHO-K1 cells were grown and assayed in DME/F-12 containing 10% FBS. CHO-K1, BHK-21 and HepG2 cells, at 25,000 cells per well, were added to a 96-well plate. After an equal volume of CellTiter-Glo ®Reagent was added, plates were shaken and luminescence monitored over time with the plates held at 22°C. The half-lives of the luminescent signals for the CHO-K1, BHK-21 and HepG2 cells were approximately 5.4, 5.2 and5.8hours, respectively.2.Product Components and Storage ConditionsProduct Size Cat.#CellTiter-Glo ®Luminescent Cell Viability Assay 10ml G7570Substrate is sufficient for 100 assays at 100µl/assay in 96-well plates or 400 assays at 25µl/assay in 384-well plates. Includes:• 1 × 10mlCellTiter-Glo ®Buffer • 1 vial CellTiter-Glo ®Substrate (lyophilized)Product Size Cat.#CellTiter-Glo ®Luminescent Cell Viability Assay 10 × 10ml G7571Each vial of substrate is sufficient for 100 assays at 100µl/assay in 96-well plates or 400 assays at 25µl/assay in 384-well plates (1,000 to 4,000 total assays). Includes:•10 × 10mlCellTiter-Glo ®Buffer •10 vials CellTiter-Glo ®Substrate (lyophilized)Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·R e l a t i v e L u m i n e s c e n c e (%)Time (minutes)CHO-K101020304050607080901003173M A 12_0AProduct Size Cat.# CellTiter-Glo®Luminescent Cell Viability Assay100ml G7572 Substrate is sufficient for 1,000 assays at 100µl/assay in 96-well plates or 4,000assays at 25µl/assay in 384-well plates. Includes:•1 × 100ml CellTiter-Glo®Buffer• 1 vial CellTiter-Glo®Substrate (lyophilized)Product Size Cat.# CellTiter-Glo®Luminescent Cell Viability Assay10 × 100ml G7573Each vial of substrate is sufficient for 1,000 assays at 100µl/assay in 96-well plates or4,000 assays at 25µl/assay in 384-well plates (10,000to 40,000 total assays). Includes:•10 × 100ml CellTiter-Glo®Buffer•10 vials CellTiter-Glo®Substrate (lyophilized)Storage Conditions:For long-term storage, store the lyophilized CellTiter-Glo®Substrate and CellTiter-Glo®Buffer at –20°C. For frequent use, the CellTiter-Glo®Buffer can be stored at 4°C or room temperature for 48hours without loss of activity. See product label for expiration date information. ReconstitutedCellTiter-Glo®Reagent (Buffer plus Substrate) can be stored at room temperaturefor up to 8hours with <10% loss of activity, at 4°C for 48hours with ~5% lossof activity, at 4°C for 4days with ~20% loss of activity or at –20°C for 21weekswith ~3% loss of activity. The reagent is stable for up to ten freeze-thaw cycles,with less than 10% loss of activity.3.Performing the CellTiter-Glo®AssayMaterials to Be Supplied by the User•opaque-walled multiwell plates adequate for cell culture•multichannel pipette or automated pipetting station for reagent delivery•device (plate shaker) for mixing multiwell plates•luminometer, CCD camera or imaging device capable of reading multiwell plates •optional:ATP for use in generating a standard curve (Section 3.C)3.A.Reagent Preparation1.Thaw the CellTiter-Glo®Buffer, and equilibrate to room temperature priorto use. For convenience the CellTiter-Glo®Buffer may be thawed andstored at room temperature for up to 48hours prior to use.2.Equilibrate the lyophilized CellTiter-Glo®Substrate to room temperatureprior to use.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3.A.Reagent Preparation (continued)3.Transfer the appropriate volume (10ml for Cat.# G7570 and G7571, or 100mlfor Cat.# G7572 and G7573) of CellTiter-Glo ®Buffer into the amber bottlecontaining CellTiter-Glo ®Substrate to reconstitute the lyophilizedenzyme/substrate mixture. This forms the CellTiter-Glo ®Reagent.4.Mix by gently vortexing, swirling or inverting the contents to obtain ahomogeneous solution. The CellTiter-Glo ®Substrate should go intosolution easily in less than 1minute.3.B.Protocol for the Cell Viability AssayWe recommend that you perform a titration of your particular cells todetermine the optimal number and ensure that you are working within thelinear range of the CellTiter-Glo ®Assay. Figure 2 provides an example of sucha titration of HEK293 cells using 0 to 50,000 cells per well in a 96-well format.1.Prepare opaque-walled multiwell plates with mammalian cells in culturemedium, 100µl per well for 96-well plates or 25µl per well for 384-wellplates.Multiwell plates must be compatible with the luminometer used.2.Prepare control wells containing medium without cells to obtain a value forbackground luminescence.3.Add the test compound to experimental wells, and incubate according toculture protocol.4.Equilibrate the plate and its contents at room temperature forapproximately 30 minutes.5.Add a volume of CellTiter-Glo ®Reagent equal to the volume of cell culturemedium present in each well (e.g., add 100µl of reagent to 100µl of mediumcontaining cells for a 96-well plate, or add 25µl of reagent to 25µl ofmedium containing cells for a 384-well plate).6.Mix contents for 2 minutes on an orbital shaker to induce cell lysis.7.Allow the plate to incubate at room temperature for 10 minutes to stabilizeluminescent signal.Note:Uneven luminescent signal within standard plates can be caused bytemperature gradients, uneven seeding of cells or edge effects in multiwellplates.8.Record luminescence.Note:Instrument settings depend on the manufacturer. An integration timeof 0.25–1 second per well should serve as a guideline.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3.C.Protocol for Generating an ATP Standard Curve (optional)It is a good practice to generate a standard curve using the same plate onwhich samples are assayed. We recommend ATP disodium salt (Cat.# P1132,Sigma Cat.# A7699 or GE Healthcare Cat.# 27-1006). The ATP standard curveshould be generated immediately prior to adding the CellTiter-Glo®Reagentbecause endogenous ATPase enzymes found in sera may reduce ATP levels.1.Prepare 1µM ATP in culture medium (100µl of 1µM ATP solution contains10–10moles ATP).2.Prepare serial tenfold dilutions of ATP in culture medium (1µM to 10nM;100µl contains 10–10to 10–12moles of ATP).3.Prepare a multiwell plate with varying concentrations of ATP standard in100µl medium (25µl for a 384-well plate).4.Add a volume of CellTiter-Glo®Reagent equal to the volume of ATPstandard present in each well.5.Mix contents for 2 minutes on an orbital shaker.6.Allow the plate to incubate at room temperature for 10 minutes to stabilizethe luminescent signal.7.Record luminescence.4.Appendix4.A.Overview of the CellTiter-Glo®AssayThe assay system uses the properties of a proprietary thermostable luciferase toenable reaction conditions that generate a stable “glow-type” luminescentsignal while simultaneously inhibiting endogenous enzymes released duringcell lysis (e.g., ATPases). Release of ATPases will interfere with accurate ATPmeasurement. Historically, firefly luciferase purified from Photinus pyralis(LucPpy) has been used in reagents for ATP assays (1,4–7). However, it hasonly moderate stability in vitro and is sensitive to its chemical environment,including factors such as pH and detergents, limiting its usefulness fordeveloping a robust homogeneous ATP assay. Promega has successfullydeveloped a stable form of luciferase based on the gene from another firefly,Photuris pennsylvanica(LucPpe2), using an approach to select characteristics thatimprove performance in ATP assays. The unique characteristics of this mutant(LucPpe2m) enabled design of a homogeneous single-reagent-addition approachto perform ATP assays with cultured cells. Properties of the CellTiter-Glo®Reagent overcome the problems caused by factors, such as ATPases, thatinterfere with ATP measurement in cell extracts. The reagent is physicallyrobust and provides a sensitive and stable luminescent output.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·4.A.Overview of the CellTiter-Glo®Assay (continued)Sensitivity and Linearity:The ATP-based detection of cells is more sensitivethan other methods (8–10). In experiments performed by Promega scientists,the luminescent signal from 50HEK293 cells is greater than three standarddeviations above the background signal from serum-supplemented mediumwithout cells. There is a linear relationship (r2= 0.99) between the luminescentsignal and the number of cells from 0 to 50,000 cells per well in the 96-wellformat. The luminescence values in Figure 2 were recorded after 10minutes ofincubation at room temperature to stabilize the luminescent signal as describedin Section3.B. Incubation of the same 96-well plate used in the experimentshown in Figure 2 for 360minutes at room temperature had little effect on therelationship between luminescent signal and number of cells (r2= 0.99).Speed:The homogeneous procedure to measure ATP using the CellTiter-Glo®Assay is quicker than other ATP assay methods that require multiple steps toextract ATP and measure luminescence. The CellTiter-Glo®Assay also is fasterthan other commonly used methods to measure the number of viable cells(such as MTT, alamarBlue®or Calcein-AM) that require prolonged incubationsteps to enable the cells’ metabolic machinery to convert indicator moleculesinto a detectable signal.4.B.Additional ConsiderationsTemperature:The intensity and decay rate of the luminescent signal from theCellTiter-Glo®Assay depends on the luciferase reaction rate. Environmentalfactors that affect the luciferase reaction rate will change the intensity andstability of the luminescent signal. Temperature is one factor that affects therate of this enzymatic assay and thus the light output. For consistent results,equilibrate assay plates to a constant temperature before performing the assay.Transferring eukaryotic cells from 37°C to room temperature has little effect onATP content (5). We have demonstrated that removing cultured cells from a37°C incubator and allowing them to equilibrate to 22°C for 1–2 hours hadlittle effect on ATP content. For batch-mode processing of multiple assayplates, take precautions to ensure complete temperature equilibration. Platesremoved from a 37°C incubator and placed in tall stacks at room temperaturewill require longer equilibration than plates arranged in a single layer.Insufficient equilibration may result in a temperature gradient effect betweenwells in the center and at the edge of the plates. The temperature gradientpattern also may depend on the position of the plate in the stack.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·Chemicals:The chemical environment of the luciferase reaction affects theenzymatic rate and thus luminescence intensity. Differences in luminescenceintensity have been observed using different types of culture media and sera.The presence of phenol red in culture medium should have little impact onluminescence output. Assaying 0.1µM ATP in RPMI medium without phenolred resulted in ~5% increase in luminescence output (in relative light units[RLU]) compared to assays in RPMI containing the standard concentration ofphenol red, whereas assays in RPMI medium containing twice the normalconcentration of phenol red showed a ~2% decrease in luminescence.Solvents for the various test compounds may interfere with the luciferasereaction and thus the light output from the assay. Interference with theluciferase reaction can be detected by assaying a parallel set of control wellscontaining medium without cells. Dimethylsulfoxide (DMSO), commonly usedas a vehicle to solubilize organic chemicals, has been tested at finalconcentrations of up to 2% in the assay and only minimally affects light output.Plate Recommendations:We recommend using standard opaque-walledmultiwell plates suitable for luminescence measurements. Opaque-walledplates with clear bottoms to allow microscopic visualization of cells also maybe used; however, these plates will have diminished signal intensity andgreater cross talk between wells. Opaque white tape may be used to decreaseluminescence loss and cross talk.Cellular ATP Content:Different cell types have different amounts of ATP,and values reported for the ATP level in cells vary considerably (1,4,11–13).Factors that affect the ATP content of cells may affect the relationship betweencell number and luminescence. Anchorage-dependent cells that undergocontact inhibition at high densities may show a change in ATP content per cellat high densities, resulting in a nonlinear relationship between cell numberand luminescence. Factors that affect the cytoplasmic volume or physiology ofcells also will affect ATP content. For example, oxygen depletion is one factorknown to cause a rapid decrease in ATP (1).Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·4.B.Additional Considerations (continued)Mixing:Optimal assay performance is achieved when the CellTiter-Glo®Reagent is mixed completely with the cultured cells. Suspension cell lines (e.g., Jurkat cells) generally require less mixing to achieve lysis and extract ATP than adherent cells (e.g., L929 cells). Tests were done to evaluate the effect ofshaking the plate after adding the CellTiter-Glo® Reagent. Suspension cellscultured in multiwell plates showed only minor differences in light outputwhether or not the plates were shaken after adding the CellTiter-Glo®Reagent.Adherent cells are more difficult to lyse and show a substantial differencebetween shaken and nonshaken plates.Several additional parameters related to reagent mixing include the force ofdelivery of CellTiter-Glo®Reagent, sample volume and dimensions of the well.All of these factors may affect assay performance. The degree of reagent mixing required may be affected by the method used to add the CellTiter-Glo®Reagent to the assay plates. Automated pipetting devices using a greater or lesser force of fluid delivery may affect the degree of subsequent mixing required.Complete reagent mixing in 96-well plates should be achieved using orbitalplate shaking devices built into many luminometers and the recommended2-minute shaking time. Special electromagnetic shaking devices that use aradius smaller than the well diameter may be required to efficiently mixcontents of 384-well plates. The depth of medium and geometry of themultiwell plates may have an effect on mixing efficiency. We recommend that you take these factors into consideration when performing the assay andempirically determine whether a mixing step is necessary for the individualapplication.LuminometersFor highly sensitive luminometric assays, the luminometer model and settings greatly affect the quality of data obtained. Luminometers from differentmanufacturers will vary in sensitivities and dynamic ranges. We recommend the GloMax®products because these instruments do not require gainadjustments to achieve optimal sensitivity and dynamic range. Additionally, GloMax®instruments are preloaded with Promega protocols for ease of use.If you are not using a GloMax®luminometer, consult the operating manual for your luminometer to determine the optimal settings. The limits should beverified on each instrument before analysis of experimental samples. The assay should be linear in some portion of the detection range of the instrument used.For an individual luminometer there may be different gain settings. Werecommend that you optimize the gain settings.4.C.References1.Crouch, S.P. et al.(1993) The use of ATP bioluminescence as a measure of cellproliferation and cytotoxicity. J. Immunol. Methods160, 81–8.2.Farfan, A.et al.(2004) Multiplexing homogeneous cell-based assays. Cell Notes10, 2–5.3.Riss, T., Moravec, R. and Niles, A. (2005) Selecting cell-based assays for drugdiscovery screening. Cell Notes13, 16–21.4.Kangas, L., Grönroos, M. and Nieminen, A.L. (1984) Bioluminescence of cellular ATP:A new method for evaluating cytotoxic agents in vitro. Med. Biol.62, 338–43.5.Lundin, A. et al.(1986) Estimation of biomass in growing cell lines by adenosinetriphosphate assay.Methods Enzymol. 133, 27–42.6.Sevin, B.U. et al.(1988) Application of an ATP-bioluminescence assay in human tumorchemosensitivity testing. Gynecol. Oncol.31, 191–204.7.Gerhardt, R.T.et al.(1991) Characterization of in vitro chemosensitivity ofperioperative human ovarian malignancies by adenosine triphosphatechemosensitivity assay. Am. J. Obstet. Gynecol. 165, 245–55.8.Petty, R.D. et al.(1995) Comparison of MTT and ATP-based assays for themeasurement of viable cell number. J. Biolumin. Chemilumin.10, 29–34.9.Cree, I.A. et al.(1995) Methotrexate chemosensitivity by ATP luminescence in humanleukemia cell lines and in breast cancer primary cultures: Comparison of the TCA-100assay with a clonogenic assay. AntiCancer Drugs6, 398–404.10.Maehara, Y. et al.(1987) The ATP assay is more sensitive than the succinatedehydrogenase inhibition test for predicting cell viability. Eur. J. Cancer Clin. Oncol.23, 273–6.11.Stanley, P.E. (1986) Extraction of adenosine triphosphate from microbial and somaticcells. Methods Enzymol.133, 14–22.12.Beckers, B. et al.(1986) Application of intracellular ATP determination in lymphocytesfor HLA-typing. J. Biolumin. Chemilumin.1, 47–51.13.Andreotti, P.E. et al.(1995) Chemosensitivity testing of human tumors using amicroplate adenosine triphosphate luminescence assay: Clinical correlation forcisplatin resistance of ovarian carcinoma. 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