BLM31PG391SN1磁珠规格书

flag磁珠说明书Anti-Flag磁珠，也称Anti-Flag免疫磁珠或Flag抗体磁珠，是由高品质的Flag 小鼠单克隆抗体与纳米级氨基磁珠共价偶联而成，可特异性地与动植物或微生物裂解液、血清、腹水等中含有Flag标签的蛋白结合，从而用于带有Flag标签的融合蛋白或其蛋白复合物的免疫沉淀(Immunoprecipitation, IP)、免疫共沉淀(Co-IP)或纯化。

Flag标签(Flag-tag)、Myc标签(Myc-tag)、HA标签(HA-tag)、His标签(His-tag)和GST标签(GST-tag)等是表达载体上最常见的一些标签，通过与这些标签的融合表达可以非常方便地检测目的蛋白及与目的蛋白相互结合的蛋白，也可以非常方便地用于目的蛋白的纯化。

Flag-tag是8个氨基酸残基(DYKDDDDK)组成的多肽，常用的形式有Flag和3X Flag，通过基因重组技术把Flag-tag的核酸序列与目的基因的5’端或3’端连接，就可以最终表达形成Flag-tag的目的蛋白。

Flag-tag具有以下优点：Flag-tag通常不会与目的蛋白相互作用，并且大多数情况下不会影响目的蛋白的功能；Flag-tag作为标签蛋白，后续通过Flag抗体(AF519)、Anti-Flag磁珠或Anti-Flag亲和凝胶(P2271)即可对目的基因的表达、定位及功能进行检测或对目的蛋白进行纯化、免疫沉淀或免疫共沉淀等；融合在N端的Flag标签，可被肠激酶(Enterokinase, EK)切除(DDDK)，从而得到完美的没有标签的目的蛋白。

基于以上优点，Flag标签已被广泛应用于蛋白表达、纯化、鉴定、相互作用和功能等多方面的研究。

Anti-Flag Magnetic Beads (Anti-Flag磁珠)，也被称为Anti-DYKDDDDK Magnetic Beads，可以特异性地结合Flag标签融合蛋白，并可以借助磁力架等磁分离设备非常便捷地应用于带有Flag标签的融合蛋白或其蛋白复合物的免疫沉淀或纯化等实验。

陶瓷贴片电容、贴片电感.片式磁珠muRuta村田命名规则与基本知识一、村田陶瓷贴片电容知识M名衣示法如卜：片状独们盹瓷电容器GRM 15.3R7K 225KE15D•GRM—农示儀锡电极：卄通貼片期瓷12容•常用的村｝ 11容就是GRM艸通贴片海瓷电容与GNM卄通贴片搏容：•18——衣示尺寸（长*宽）:1.6*0.8mm即内通用尺对衣示是（K*审）1.6*0.8mm （单位为mm：c词阿卜通用尺、J A示是用英寸0603 （单位为inch；，卜J E询常用代码仃03、15、18、21、31、32、42、43、55〕；，II 体的对应值如卜I03-...0.6*0.3mm- (0201)15——1.0*0.5mm~-040218-...1.6*0.8mm- (0603)21-—2.0# 1.25mm——080531一“3.2* 1.6mm—120632-—3.2* 1.5mm一・121042一.5*2.0mm——180843 …45*3・2mm•—181255—5.7 拿5・0mm——2220•8——衣示用度（T：: 0.8mm常用厚度村田代码仃5、6、8、9、B、C、E：'h貝㈱对应值如卜：5-…0.5mm 6-…0.6mm 8-…0.8mm 9-…0.9mm B-…1.25mm C-…1.6mm E -—2.5mm•R7——农示材质：X7R常用材质村t：代码有5C、R6、R7、F5等,R体的对应值如卜：5C--COG/NPO/CHR6——X5RR7——X7RF5・——Y5V5C I仆温度是・55度一+125必温度系敌是0+・30ppm/gR6 I作泪度是・55度一H85度，SH度系数是+-15%:R7 1作温度是・55度一H25度,温度系数好+15%:F5 I作fiU是・30度—85度.温度系数是+22 -82%lOOpfW卜小容fi*L般采比5C材頂，100PF—luf人一般采用R7材质，luf以上一股采用R6材质,櫛S®求不禹的般采用F5材质。

提供村田磁珠规格书村田电容规格书村田电感规专业销售日本村田MURATA品牌电子元器件.村田电容,村田贴片电容,村田陶瓷电容,村田安规电容,村田可调电容,村田电感,村田贴片电感,村田绕线电感,村田叠层电感,村田高频电感,村田功率电感,村田屏蔽电感,村田薄膜电感,村田磁珠,村田贴片磁珠,村田EMI磁珠,村田EMI滤波器,村田声表滤波器,村田蓝牙滤波器,村田陶瓷振荡子.村田三端子滤波器,村田热敏电阻,村田NTC热敏电阻,村田贴片热敏电阻.村田射频头,村田射频测试线,村田RF同轴连接器,村田传感器等等.并提供相应产品规格书.选型指南.村田代理现货供应日本村田MURATA 电子元器件贴片陶瓷电容常用料如下：GRM155R61C104KA88D、GRM155R61A105KE15D、GRM155R60J225ME15D 、GRM185R61C105KE44DGRM188R61C105KA93D、GRM188R60J225KE19D 、GRM188R61A225KE34D 、GRM188R60J475KE19DGRM188R71A225KE15D、GRM188R60J106ME47D 、GRM21BR71C105KA01L、GRM219R60J106KE19DGRM21BR60J106KE19L、GRM219R60J226ME47D 、GRM21BR60J226ME39L、GRM31CR61E106KA12LGRM32ER61C476KE15L、GRM32ER60J107ME20L、GNM214R61A105ME17D、GNM1M2R61C105ME18D、GNM1M2R61A105JNE17D、TZC3Z060A110R00 TZC3R100A110R00 TZV2R200A110 TZV2R030A110 TZV2Z06 0A110TZY2Z060A001 TZY2Z100A001 TZY2Z200A001电感常用料如下；LQG15HS1N5S02、LQG15HN15NJ02、LQG15HN18NJ02、LQG15HN3N9S02、LQG15HS5N6S02、LQG15HS6N8J02、LQP15MN8N2B02、LQP15MN4N7B02D、LQP15MN5N6B02D、LQP15MN27NJ02D、LQP18MN15NG02D、LQP18MN2N7C02D、LQP18MN3N3C02D、LQP18MN1N8C02、LQP18MN4N7C02D、LQP18MN27NG02D、LQH31MN100K03L、LQW15AN39NG00D、LQW15AN47NG00D、LQH43MN102K03L、LQW15AN5N6C10D、LQW15AN2N2C10D、磁珠常用料如下：BLM18HD102SN1D、BLM15BB750SN1D、BLM18BD252SN1D、BLM15BD182SN1D、BLM18PG221SN1D、BLM18HG601SN1D、BLM21AG121SN1D、BLM21PG221SN1D、BLM31PG330SN1L、BLM15HG601SN1D、BLM15AG121SN1D、BLM21BB331SN1D、BLM31PG601SN1L、BLM41PG600SN1L、BLM15BD102SN1D、BLM18HD102SN1D、热敏电阻常用料如下：NCP15WF104F03RC NCP21WB473J03RA NCP21XM472J03RA NCP21XW223J03RA NCP15XC220J03RC NCP15XW682J03RCNCP15XQ102J03RC NCP21XV103J03RA NCP15WB333J03RA NCP18XQ102J03R B NCP15XH103F03RC NCP18XH103F03RB陶瓷振荡子常用料如下：CSTCE16M0V53-R0、CSTCR4M00G53-R0、CSTCR6M00G53-R0、CSTLS20M0X53-B0、CSTCE18M4V53-R0、CSTCE8M00G52-R0、CSTLS4M00G53-B0、蓝牙滤波器常用料如下：LFB182G45SG9A293、LFB212G45BA1A220、LFB212G45SG8A192、声表滤波常用料如下：SAWEP942MCM0F00R12、SAFEA1G96FA0F00R12、SAFEA942MFA0F00、SAFEA881MAA0F00、SAFEA1G96AB0F00、SAFEA942MFL0F00R14、SAFEA1G84FA0F00R14、SAFEA881MFL0F00R14、SAWEN942MCM0F00R14、SAFEB1G57FM0F00、SAFEL1G96FA0F00R14、SAFEL1G84FA0F00R14、SAFEL942MFL0F00R14、SAFEL881MFL0F00R14滤波电容常用料号如下：NFM21PC105B1A3、NFM3DPC223R1H3L、NFE31PT221D1E9L、NFM18CC223R1C3D、NFM18PC105R0J3、NFM21PC474R1C3D、NFM3DCC101U1H3L、NFM41PC204F1H3L、NFM21PC104R1E3D、NFM21CC223R1H3D、NFM21PC224R1C3D、NFM3DCC471R1H3、NFM21CC222R1H3B、NFE31PT222Z1E9、NFE61PT101Z1H9L、NFE61PT472C1H9L、NFW31SP506X1E4、NFM41PC204F1H3、NFM41CC223R2A3L、NFM18PC104R1C3B、NFM18PC105R03J、BNX002-01共模扼流线圈常用料如下：DLW21SN900SQ2L、DLW21SN670SQ2L、DLW5BTN102SQ2L、DLP31SN221SL2、DLW5BTN142SQ2L、DLW5BSN152SQ2L、DLW5BSN191SQ2L、DLW5BTN101SQ2L、DLW5BSN302SQ2L、DLP31SN121ML2L、DLP11SN121SL2、DXW21BN7511T、DXP18BN5014TL射频元件常用料如下：射频测试线MXHS83QE3000 射频探头手机测试头MM126036 MM126038 同轴连接器 MM8430-2610 MM9329-2700天线开关常用料如下：LMSP33CA-465、LMSP33AA-696 、LMSP33AA-298、LMSP33QA-382、LMSP43MA-506联系人：朱先生手机：136******** QQ：1084623702 MSN：****************常用邮箱：*****************备用邮箱：****************公司地址：深圳市福田区上步南路国企大厦永富楼23F。

PRODUCT DETAIL Electrical CharacteristicsTest InstrumentsZ Ω (Ref. Page 6~8) DCR Ω(Ref. Page 6~8) IDCmA(Ref. Page 6~8) TEST FREQ: MHz(Ref. Page 6~8)TEST LEVEL: 250 mV•HP4291BRF IMPEDANCE / MATERIAL ANALYZER •HP4338A/B MILLIOHMMETER •Agilent 8720ES S-PARAMETER NETWORK ANALYZER•HP6632B SYSTEM DC POWER SUPPLYPART NUMBER CODEMHC 1608 S 12 1 P B P 1 2 3 4 5 6 7 8 1 Series Name2 Size Code: the first two digitals : length(mm), the last two digitals : width(mm)3 Product Characteristics : S = For Standard4 Impedance(Ω) ± 25%5 Fixed Decimal Point6 Rated Current Code7 Soldering : Green Parts: A— Soldering Lead-Free B— Lead-Free for whole chip 8 Packaging: P - Embossed paper tape, 7" reel. E - Embossed plastic tape, 7" reel.L =1000mA M =1500mA N =2000mA P =2500mAQ =3000mA R =4000mA U =5000mA W =6000mA (ex ：600=60Ω ；121=120Ω)TAPE AND REEL SPECIFICATIONSPLASTIC CARRIERPAPER CARRIERTAPING DIMENSIONSUnit: mmSize4532 4516 3225 3216 2012 1608 1005Symbol PLASTIC PLASTIC PLASTIC PLASTICPAPER PAPER PAPER W12.0±0.10 11.7~12.3 7.70~8.307.90~8.308.00±0.108.00±0.108.00±0.10 P 8.00±0.10 4.00±0.10 4.00±0.10 4.00±0.10 4.00±0.10 4.00±0.10 2.00±0.05 E 1.75±0.10 1.75±0.10 1.75±0.10 1.75±0.10 1.75±0.10 1.75±0.10 1.75±0.05 F 5.50±0.05 5.50±0.05 3.50±0.05 3.50±0.05 3.50±0.10 3.50±0.10 3.50±0.05 D1.55±0.05 1.55±0.05 1.55±0.05 1.55±0.051.56±0.10 1.56±0.10 1.55±0.05 D1 1.50~1.75 1.50~1.75 0.95~1.200.95~1.20NA NA NA Po 4.00±0.10 4.00±0.10 4.00±0.10 4.00±0.10 4.00±0.10 4.00±0.104.00±0.10Po10 40.0±0.20 40.0±0.20 40.0±0.2040.0±0.2040.0±0.20NA NAP2 2.00±0.05 2.00±0.05 2.00±0.05 2.00±0.05 2.00±0.10 2.00±0.10 2.00±0.05 Ao 3.66±0.10 1.83±0.10 2.57±0.10 1.85±0.10 1.50±0.05 1.05±0.050.62±0.03 Bo4.95±0.10 4.85±0.10 3.40±0.10 3.43±0.102.30±0.05 1.85±0.05 1.12±0.03 Ko(T) 1.83±0.10 1.83±0.10 1.32±0.10 1.22±0.100.95±0.050.95±0.050.60±0.03t0.23±0.10 0.29±0.10 0.25±0.100.25±0.10NA NA NAREEL DIMENSIONSUnit: mmReel Packaging Quantity PART SIZE (EIA SIZE) 1005 (0402)1608 (0603)2012 (0805)3216 (1206)3225 (1210)4516 (1806)4532 (1812)7” REELQty. (pcs)10,000 4,000 4,000 3,000 2,000 2,000 1,000The Contents of a box :4532(1812): 4 reels / inner box; 4516(1806): 4 reels / inner box; 3225(1210): 5 reels / inner box; 3216(1206): 5 reels / inner box; 2012(0805): 5 reels / inner box; 1608(0603): 5 reels / inner box; 1005(0402): 5 reels / inner box.RECOMMENDED SOLDERING CONDITIONSLAND PATTERNS FOR REFLOW SOLDERINGSOLDER LAND INFORMATION Unit: mm (inches)RELIABILITY AND TEST CONDITIONTest itemTest conditionCriteriaTemperature Cyclea. Temperature : -40 ~ +85℃b. Cycle : 100 cyclesc. Dwell time : 30minutesd.Measurement : at ambient temperature 24 hrs after test completiona. No mechanical damageb. Impedance value should be within ± 20 % of the initial valueOperational Lifea. Temperature : 125℃ ± 5℃b. Test time : 1000 hrsc. Apply current : full rated currentd.Measurement : at ambienttemperature 24 hrs after testcompletiona. No mechanical damageb. Impedance value should bewithin ± 20 % of the initialvalueBiased Humiditya. Temperature : 40℃ ± 2℃b. Humidity : 90 ~ 95 % RHc. Test time : 1000 hrsd. Apply current : full rated currente.Measurement : at ambient temperature 24 hrs after test completiona. No mechanical damageb. Impedance value should be within ± 20 % of the initial valueSize A B C0.4 ～ 0.6 1.6 ～ 2.6 0.4 ～ 0.7 1005 (0.015 ～ 0.023) (0.063 ～ 0.102) (0.016 ～ 0.027) 0.5 ～ 0.7 2.1 ～ 3.1 0.65 ～ 0.95 1608 (0.019 ～ 0.027) (0.083 ～ 0.122) (0.026 ～ 0.037) 1.0 ～ 1.2 3.0 ～ 4.0 0.8 ～ 1.1 2012 (0.039 ～ 0.047) (0.118 ～ 0.157) (0.031 ～ 0.043) 2.0 ～ 2.4 4.2 ～ 5.2 1.0 ～ 1.4 3216 (0.079 ～ 0.094) (0.165 ～ 0.204) (0.039 ～ 0.055) 2.1 ～ 2.3 4.2 ～ 5.2 2.2 ～ 2.5 3225 (0.082 ～ 0.090) (0.165 ～ 0.204) (0.086 ～ 0.098) 3.4 ～ 3.7 6.3 ～ 7.3 1.3 ～ 1.7 4516 (0.133 ～0.145) (0.248 ～ 0.287) (0.051 ～ 0.067) 3.4 ～ 3.7 6.3 ～ 7.3 2.9 ～ 3.2 4532(0.133 ～0.145) (0.248 ～ 0.287)(0.144 ～ 0.126)Resistance to Solder Heat a. Solder temperature : 260 ± 5℃b. Flux : Rosinc. DIP time : 10 ± 1 seca. More than 95 % of terminal electrode should be covered with new solderb. No mechanical damagec. Impedance value should be within ± 20 % of the initial valueAdhesive Testa. Reflow temperature : 245℃ It shall be Soldered on thesubstrate applying direction paralle1 to the substrateb. Apply force(F) : 5 Nc. Test time : 10 seca. No mechanical damageb. Soldering the products on PCB after the pulling test force > 5 NGENERAL TECHNICAL DATAOperating temperature range : - 55℃ ~ +125℃ Storage Condition : Less than 40℃ and 70% RH Storage Time: 6 months(Size:1005) 12 months(Size:1608 above) Soldering method: Reflow or Wave SolderingTest itemTest condition CriteriaSteam Aging Testa. Temperature : 93℃b. Test time : 4 hrs(MHC1005)Others : 8 hrsc. Solder temperature : 235 ± 5℃d. Flux : Rosine. DIP time : 5 ± 1 secMore than 95 % of terminal electrode should be covered withnew solder Rated Current Testa. Apply current : full ratedcurrent / 5minTemperature rise should be less than 40℃PART NUMBER AND CHARACTERISTICS TABLEImpedance (Ω)+/-25% Test Freq. (MHz) DCR(Ω) (Max.) Rated Current(mA)MHC1005 SeriesMHC1005S100NB_ 10 100 0.09 2000 MHC1005S300NB_ 30 100 0.09 2000 MHC1005S600LB_ 60 100 0.2 1000 MHC1005S121MB_ 120 100 0.15 1500 MHC1608 SeriesMHC1608S300QB_ 30 100 0.04 3000 MHC1608S600QB_ 60 100 0.04 3000 MHC1608S800QB_ 80 100 0.04 3000 MHC1608S121PB_ 120 100 0.07 2500 MHC1608S221NB_ 220 100 0.09 2000 MHC1608S301NB_ 300 100 0.09 2000 MHC1608S471LB_ 470 100 0.20 1000 MHC1608S601LB_ 600 100 0.20 1000 MHC2012 SeriesMHC2012S110WB_ 11 100 0.015 6000 MHC2012S310WB_ 31 100 0.015 6000 MHC2012S400RB_ 40 100 0.03 4000 MHC2012S600QB_ 60 100 0.04 3000 MHC2012S800UB_ 80 100 0.02 5000 MHC2012S900UB_ 90 100 0.02 5000 MHC2012S121UB_ 120 100 0.02 5000 MHC2012S221QB_ 220 100 0.04 3000 MHC2012S251QB_ 250 100 0.04 3000 MHC2012S301NB_ 300 100 0.09 2000 MHC2012S331NB_ 330 100 0.09 2000 MHC2012S601NB_ 600 100 0.09 2000Impedance (Ω)+/-25%Test Freq. (MHz)DCR(Ω) (Max.)Rated Current(mA)MHC3216 SeriesMHC3216S300WB_ 30 100 0.015 6000 MHC3216S500WB_ 50 100 0.015 6000 MHC3216S600QB_ 60 100 0.04 3000 MHC3216S750QB_ 75 100 0.04 3000 MHC3216S800RB_ 80 100 0.03 4000 MHC3216S900QB_ 90 100 0.04 3000 MHC3216S121WB_ 120 100 0.015 6000 MHC3216S151NB_ 150 100 0.09 2000 MHC3216S501PB_ 500 100 0.07 2500 MHC3216S601PB_ 600 100 0.07 2500 MHC3216S122LB_ 1200 100 0.2 1000 MHC3225 SeriesMHC3225S600MB_ 60 100 0.15 1500 MHC3225S102NB_ 1000 50 0.09 2000 MHC4516 seriesMHC4516S600WB_ 60 100 0.015 6000 MHC4516S471NB_ 470 100 0.09 2000 MHC4516S851MB_ 850 100 0.15 1500 MHC4532 seriesMHC4532S800WB_ 80 100 0.015 6000 MHC4532S121WB_ 120 100 0.015 6000 MHC4532S601QB_ 600 50 0.04 3000 MHC4532S681QB_ 680 50 0.04 3000 MHC4532S132QB_ 1300 60 0.04 3000** For special part number which is not shown in the above table, please refer to appendix.PART COMPOSITION DECLARATION。

Contents1. Description1.1 Principle of the MACS® Separation1.2 Background information1.3 Applications1.4 Reagent and instrument requirements试剂和仪器的要求2. Protocol2.1 Sample preparation样品制备2.2 Magnetic labeling磁性标记2.3 Magnetic separation磁性分离3. Example of a separation using the CD133 MicroBead Kit4. References1. DescriptionComponents 2 mL CD133 MicroBeads, human:MicroBeads conjugated to monoclonal antihumanCD133 antibodies (isotype: mouse IgG1,clone AC133).2 mL FcR Blocking Reagent, human Specificity CD133 antigen, epitope (CD133/1)1.Capacity For 2亊10⁹total cells, up to 100 separations.Product format CD133 MicroBeads are supplied in buffercontaining stabilizer and 0.05% sodium azide.Storage Store protected from light at 2−8 °C. Do not freeze. The expiration date is indicated on the vial label.1.1 Principle of the MACS® SeparationFirst, the CD133+ cells are magnetically labeled with CD133MicroBeads. Then, the cell suspension is loaded onto a MACS®Column, which is placed in the magnetic field of a MACS Separator.The magnetically labeled CD133+ cells are retained within thecolumn. The unlabeled cells run through; this cell fraction isthus depleted of CD133+ cells. After removing the column fromthe magnetic field, the magnetically retained CD133+ cells can beeluted as the positively selected cell fraction. To increase the purity,the positively selected cell fraction containing the CD133+ cells isseparated over a second column.1.2 Background informationThe CD133 MicroBead Kit is a magnetic labeling system designedfor the positive selection of CD133+ cells. It allows the single-stepisolation of nonhematopoietic and early hematopoietic progenitors and stem cells. The CD133 molecule is a 5-transmembrane cellsurface antigen with a molecular weight of 117 kD.2 The CD133/1(clone AC133) antibody recognizes epitope 1 of the CD133 antigen.1In the hematopoietic system, CD133 expression is restricted to asubset of CD34bright stem and progenitor cells in human fetal liver,bone marrow, cord blood, and peripheral blood.3 Isolated fromhematopoietic sources, CD133+ cells can become adherent and arereported to become CD133–during culture⁴. The CD34+CD133+cell population, which includes CD34+CD38–cells, was shown tobe capable of repopulating NOD/SCID mice.⁵Recently, CD133has also been found to be expressed on circulating endothelialprogenitor cells⁶,⁷and fetal neural stem cells⁸,⁹as well as on othertissue-specific stem cells, such as renal1⁰and prostate11 stem cells.Lately, when isolated from tumor tissue, the CD133+ populationcan be enriched for tumor-initiating cells.11-1⁵CD133 MicroBeadshave been used to isolate adult stem cells from cord blood and asa starting population for reprograming towards iPS cells.1⁶CD133expression has been found on undifferentiated human ES cells.Therefore CD133 MicroBeads could be used for enrichment ordepletion of these cells.1⁷1.3 Applications●Positive selection or depletion of cells expressing human CD133antigen.●Isolation or depletion of CD133+ cells from peripheral bloodmononuclear cells (PBMCs) or single-cell suspensions fromtissue.●CD133+ cells are used in basic stem cell research, stem cellevaluation, stem cell expansion, research in hematologicalmalignancies, stem cell plasticity, and potential cellulartherapies as well as in tissue regeneration and cancer research.1.4 Reagent and instrument requirements●Buffer: Prepare a solution containing phosphate-buffered saline(PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mMEDTA by diluting MACS BSA Stock Solution (# 130‑091-376)1:20 with autoMACS Rinsing Solution (# 130-091-222). Keepbuffer cold (2−8 °C). Degas buffer before use, as air bubblescould block the column.▲Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.●(Optional) Fluorochrome-conjugated antibodies forflow cytometric analysis, e.g., CD133/2 (293C3)-PE(# 130-090-853), CD133/2 (293C3)-APC (# 130-090-854),CD133/2 (293C3)‑Biotin, CD34-FITC (# 130-081-001),CD34‑APC (# 130-090-954), or CD34-PE (# 130-081-002). Formore information about fluorochrome-conjugated antibodiessee .●MACS Columns and MACS Separators: CD133+ cells can beEnriched浓缩by using MS, LS, or XS Columns or depleted withthe use of LD, CS, or D Columns. Cells which strongly expressthe CD133 antigen can also be depleted using MS, LS, or XSColumns. Positive selection正向or depletion can also be performedby using the autoMACS Pro or the autoMACS Separator.▲Note: Column adapters are required to insert certain columns into theV arioMACS™or SuperMACS™Separators. For details see the respective MACS Separator data sheet.●(Optional) Propidium Iodide Solution (# 130-093-233) or7-AAD for flow cytometric exclusion of dead cells.●(Optional) Dead Cell Removal Kit (# 130-090-101) for thedepletion of dead cells.●(Optional) Pre-Separation Filters (# 130-041-407) to removecell clumps团.2. Protocol2.1 Sample preparationWhen working with anticoagulated peripheral blood or buffy coat,peripheral blood mononuclear cells (PBMCs) should be isolated bydensity gradient centrifugation, for example, using Ficoll-Paque™.▲Note: To remove platelets after density gradient separation, resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully aspirate supernatant. Repeat washing step.When working with tissues or lysed blood, prepare a single-cellsuspension using standard methods.For details see the protocols section at /protocols.For preparation of cord blood cells, bone marrow cells, or cellsfrom leukapheresis material, please refer to the sample preparationprotocols at /protocols.▲Dead cells may bind non-specifically to MACS MicroBeads.To remove dead cells, we recommend using density gradientcentrifugation or the Dead Cell Removal Kit (# 130-090-101).2.2 Magnetic labeling▲Work fast, keep cells cold, and use pre-cooled solutions. This willprevent capping of antibodies on the cell surface and non-specificcell labeling.▲V olumes for magnetic labeling given below are for up to10⁸total cells. When working with fewer than 10⁸cells, use the same volumes as indicated. When working with higher cell numbers,scale up all reagent volumes and total volumes accordingly (e.g.for 2亊10⁸total cells, use twice the volume of all indicated reagent volumes and total volumes).▲For optimal performance it is important to obtain a single‑cell suspension before magnetic labeling. Pass cells through 30 μm nylonmesh (Pre-Separation Filters, # 130-041-407) to remove cell clumpswhich may clog the column. Moisten filter with buffer before use.▲The recommended incubation temperature is 2–8 °C. Workingon ice may require increased incubation times. Higher temperaturesand/or longer incubation times may lead to non-specific celllabeling.1. Determine确定cell number.2. Centrifuge cell suspension细胞悬液at 300×g for 10 minutes. Aspirate supernatant completely.吸取上清3. Resuspend cell pellet in 300 μL of buffer per 10⁸total cells.4. Add 100 μL of FcR Blocking Reagent per 10⁸total cells.5. Add 100 μL of CD133 MicroBeads per 10⁸total cells.6. Mix well and incubate for 30 minutes in the refrigerator(2−8 °C).7. (Optional) Add staining antibodies, e.g., 50 μL of CD133/2(293C3)-PE (# 130-090-853), and incubate for 5 minutes in thedark in the refrigerator (2−8 °C).8. Wash cells by adding 1−2 mL of buffer per 10⁸cells andcentrifuge at 300×g for 10 minutes. Aspirate supernatantcompletely.9. Resuspend up to 10⁸cells in 500 μL of buffer.▲Note: For higher cell numbers, scale up buffer volume accordingly.▲Note: For depletion with LD Columns, resuspend up to 1.25亊10⁸cells in 500 μL of buffer.10. Proceed to magnetic separation (2.3).2.3 Magnetic separation▲Choose an appropriate MACS Column and MACS Separatoraccording to the number of total cells and the number of CD133+cells. For details see table in section 1.4.▲Always wait until the column reservoir is empty before proceedingto the next step.Magnetic separation with MS or LS Columns▲To achieve highest purities, perform two consecutive columnruns.1. Place column in the magnetic field of a suitable MACS Separator.For details see the respective MACS Column data sheet.2. Prepare column by rinsing with the appropriate amount ofbuffer:MS: 500 μL LS: 3 mL3. Apply cell suspension onto the column. Collect flow-throughcontaining unlabeled cells.4. Wash column with the appropriate amount of buffer. Collectunlabeled cells that pass through and combine with the effluentfrom step 3.MS: 3×500 μL LS: 3×3 mL▲Note: Perform washing steps by adding buffer aliquots only when the column reservoir is empty.5. Remove column from the separator and place it on a suitablecollection tube.▲Note: To perform a second column run, you may elute the cells directly from the first onto the second, equilibrated column instead of a collection tube.6. Pipette the appropriate amount of buffer onto the column.Immediately flush out the magnetically labeled cells by firmlypushing the plunger into the column.MS: 1 mL LS: 5 mL7. To increase purity of CD133+ cells, enrich the eluted fractionover a second MS or LS Column. Repeat the magneticseparation procedure as described in steps 1 to 6 by using anew column.Magnetic separation with XS ColumnsFor instructions on the column assembly and the separation refer tothe XS Column data sheet.Depletion with LD Columns1. Place LD Column in the magnetic field of a suitable MACSSeparator. For details see LD Column data sheet.2. Prepare column by rinsing with 2 mL of buffer.3. Apply cell suspension onto the column.4. Collect unlabeled cells that pass through and washcolumn with 2×1 mL of buffer. Collect total effluent;this is the unlabeled cell fraction. Perform washingsteps by adding buffer two times. Only add new buffer whenthe column reservoir is empty.Depletion with CS Columns1. Assemble CS Column and place it in the magnetic field of asuitable MACS Separator. For details see CS Column datasheet.2. Prepare column by filling and rinsing with 60 mL of buffer.Attach a 22G flow resistor to the 3-way stopcock of theassembled column. For details see CS Column data sheet.3. Apply cell suspension onto the column.4. Collect unlabeled cells that pass through and wash columnwith 30 mL buffer from the top. Collect total effluent; this isthe unlabeled cell fraction.Depletion with D ColumnsFor instructions on column assembly and separation refer to theD Column data sheet.Magnetic separation with the autoMACS® Pro Separator or theautoMACS® Separator▲Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator or the autoMACS Separator.▲Buffers used for operating the autoMACS Pro Separator or the autoMACS Separator should have a temperature of ≥10 °C.▲Program choice depends on the isolation strategy, the strengthof magnetic labeling, and the frequency of magnetically labeled cells. For details refer to the section describing the cell separation programs in the respective user manual.Magnetic separation with the autoMACS® Pro Separator1. Prepare and prime the instrument.2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Placesample tube in row A of the tube rack and the fractioncollection tubes in rows B and C.3. For a standard separation choose one of the following programs:Positive selection from peripheral blood, bone marrow, or leukapheresis: “Posseld”Collect positive fraction in row C of the tube rack.Positive selection from cord blood: “Posseld2”Collect positive fraction in row C of the tube rack.Depletion: “Depletes”Collect negative fraction in row B of the tube rack.Magnetic separation with the autoMACS® Separator1. Prepare and prime the instrument.2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Placesample tube at the uptake port and the fraction collectiontubes at port neg1 and port pos 2.3. For a standard separation choose one of the following programs:Positive selection from peripheral blood, bone marrow, or leukapheresis: “Posseld”Collect positive fraction from outlet port pos 2.Positive selection from cord blood: “Posseld2”Collect positive fraction from outlet port pos 2.Depletion: “Depletes”Collect negative fraction from outlet port neg1.3. Example of a separation using the CD133MicroBead KitCD133+ hematopoietic stem and progenitor cells were isolated from non-mobilized human PBMCs using the CD133 MicroBead Kit, MS Columns, and a MiniMACS™Separator. Cells were fluorescently stained with CD34-FITC (# 130-081-001) and CD133/2 (293C3)-PE (# 130-090-853) and analyzed by flow cytometry. Cell debris and dead cells werde excluded from the analysis based on scatter signals and propidium iodide fluorescence.All protocols and data sheets are available at .WarningsReagents contain sodium azide. Under acidic conditions sodium azide yields hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with running water before discarding. These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop.WarrantyThe products sold hereunder are warranted only to be free from defects in workmanship and material at the time of delivery to the customer. Miltenyi Biotec GmbHmakes no warranty or representation, either expressed or implied, with respect tothe fitness of a product for a particular purpose. There are no warranties, expressedor implied, which extend beyond the technical specifications of the products.Miltenyi Biotec GmbH’s liability is limited to either replacement of the products or refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property damage, personal injury or economic loss caused by the product.autoMACS and MACS are registered trademarks and MidiMACS, MiniMACS, OctoMACS, QuadroMACS, SuperMACS, and V arioMACS are trademarks of Miltenyi Biotec GmbH.Ficoll-Paque is a trademark of GE Healthcare companies.Copyright . 2009 Miltenyi Biotec GmbH. All rights reserved.。