_-_-Blebbistatin_DataSheet_MedChemExpress

Inhibitors, Agonists, Screening Libraries Data SheetBIOLOGICAL ACTIVITY:Mubritinib(TAK–165) is a potent and selective inhibitor of HER2/ErbB2 with IC50 of 6 nM; with no inhibition on EGFR, FGFR, PDGFR etc.IC50 value: 6 nM [1]Target: HER2 inhibitorin vitro: Mubritinib displays > 4000–fold selectivity over other tyrosine kinases, such as EGFR, FGFR, PDGFR, Jak1, Src and Blk.Mubritinib even at low concentration of 0.1 μM significantly blocks HER2 phosphorylation, leading to the downregulation of PI3K–Akt and MAPK pathway in cell line BT474 with high level of HER2. Mubritinib not only exhibits highly potent antiproliferative effect in ErbB2–overexpressing cancer cell line BT474 with an IC50 of 5 nM, but also displays marked antiproliferative effects in cell lines with HER2 expressed weakly with IC50 of 53 nM, 90 nM and 91 nM for LNCaP, LN–REC4 and T24, respectively. Mubritinib displays no inhibitory activities against PC–3 cells with HER2 expressed very faintly with IC50 of 4.62 μM, as well as EGFR–overexpressing HT1376and ACHN cell lines with IC50 of >25 μM [1].in vivo: Mubritinib significantly inhibits LN–REC4 xenograft with treatment/control tumor volume ratio of 26.5%. Although ineffective to inhibit the growth of UMUC–3 and ACHN cells in vitro (IC50s of 1.812 and >25 μM, respectively), oral administration of Mubritinib (10 or 20 mg/kg per day) significantly inhibits the growth of UMUC–3 and ACHN xenografts with treatment/control tumor volume ratio of 22.9% and 26%, respectively, as compared with Herceptin (20 mg/kg) which is ineffective to UMUC–3 tumor growth [1].PROTOCOL (Extracted from published papers and Only for reference)Cell assay [1]:To determine HER2/erbB2 tyrosine kinase phosphorylation, BT–474 cells (2 × 105 cells/well) were seeded on 24–well plates andcultured overnight. TAK–165 was then added at various concentrations. After incubation for 2 h, the cells were harvested directly into sodium dodecyl sulfate (SDS)–sample buffer (200 μL). To determine other receptor–type tyrosine kinase inhibition, A–431 cells or NIH3T3 fibroblasts (2 × 105 cells/well) were seeded in medium containing 10% FBS and incubated overnight to allow the cells to adhere to the plates. The medium was replaced with serum–free medium, and the cells were allowed to grow for another day.TAK–165 was added 2 h before the addition of growth factors (A–431cells: EGF [20 ng/mL]; NIH3T3 fibroblasts: PDGF [20 ng/mL] or FGF [20 ng/mL]). After incubation for 5 min, the cells were lyzed in SDS–sample buffer.Xenograft studies in mice [1]:UMUC–3 and ACHN cells were implanted subcutaneously to nude mice. Castration was performed on SCID mice 1 week before implantation of LN–REC4 cells. UMUC–3 and LN–REC4 cells were implanted with 50% Matrigel solution (Becton Dickinson, Franklin Lakes, NJ, USA). After the tumor volume reached 200–300 mm3 in LN–REC4 and UMUC–3 cells and to 100–200 mm3 in ACHN, the mice were treated orally twice daily for 14 days with vehicle (control) or 10 or 20 mg/kg per day of TAK–165. In the Herceptin study against UMUC–3, treatments consisted of a twice weekly intraperitoneal injection of 20 mg/kg Herceptin in PBS for 2 weeks. TumorProduct Name:Mubritinib Cat. No.:HY-13501CAS No.:366017-09-6Molecular Formula:C 25H 23F 3N 4O 2Molecular Weight:468.47Target:EGFR; EGFR Pathway:JAK/STAT Signaling; Protein Tyrosine Kinase/RTK Solubility:10 mM in DMSOgrowth was assessed by electronic caliper measurement of tumor diameter in two dimensions, and tumor volume was calculated using the equation: tumor volume = A × B2 × 1/2 (mm3) where A is the largest diameter of the tumor and B is the smallest diameter of the tumor. Tumor size was measured every 2 days throughout the study period. The antitumor effect was expressed as growth of each treated tumor/average growth of control tumor × 100 [T/C (%)].References:[1]. Nagasawa J, et al. Novel HER2 selective tyrosine kinase inhibitor, TAK–165, inhibits bladder, kidney and androgen–independent prostate cancer in vitro and in vivo. Int J Urol. 2006 May;13(5):587–92.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

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(±)-Blebbistatin 是一种细胞可透过性的非肌肉肌球蛋白 II 型 ATP 酶抑制剂,IC50 值为 2 μM [1,2]. 非肌肉肌球蛋白 II(NM II)是一种肌动蛋白结合蛋白,在调节细胞迁移\黏附及分化中发挥主要 作用[4].用基因缺失和突变的方法更加证实了 NM II 在这些过程中的重要性,NM II 突变影响了 大范围的蛋白功能,且引发单基因病[5.6].
(±)-Blebbistatin 是一种小分子抑制剂,与肌球蛋白-ADP-Pi 复合体优先结合以放慢磷酸盐的释 放[2].该抑制剂完全消除肌动蛋白激活的 Mg-ATP 酶的收缩活性和体外几种物种的肌球蛋白 II 的运动性(IC50 = 0.5-5.0 μM) [8,9],但是,它对平滑肌肌球蛋白 II (IC50 =80 μM)\肌球蛋白 I\V 及 X 的效果差[3].此外,blebbistatin 能够强效抑制哺乳动物动脉平滑肌(IC50=5 μM) [9].在体 内应用中,blebbistatin 所具有的阻滞处于肌动蛋白分离状态的肌球蛋白 II 及阻止刚性肌动球 蛋白交联的特性是一个巨大优势[2,11]. 在恒压灌注模型系统中,CB 和 TM 细胞用 blebbistatin (10-200 M)处理后,细胞形态有所变化, 肌动蛋白应力纤维含量减少.经过 24 小时的冲刷,Blebbistatin 效果是完全可逆的 [10].Blebbistatin 抑制单细胞收缩而不改变细胞内钙瞬变的形态(IC50 = 0.43 μM).暴露于波 长小于 488 nm 的紫外光下也可迅速抑制 blebbistatin[8].

Becton, Dickinson and Company BD Biosciences2350 Qume Drive San Jose, CA 95131 USA 5/201523-6755-03***************************For Research Use Only. Not for use in diagnostic or therapeutic procedures.••••••••••••Live and Dead Cell Discrimination BD™ Cell Viability Kit Catalog No.Number of tests 349483100 Tests 349480 with BD Liquid Counting Beads 100 Tests RESEARCH APPLICATIONS Studies of:•rapid counting of live/dead bacteria or other microbial cells 1-4 •efficacy of bacterial disinfectant 5 •viability of yeast during fermentation 6 •viability and concentration of cells in culture •viability and concentration of cells before staining for flow cytometric analysis •mammalian or microbial cells •viability and count of cells in bioreactors •viability of sperm for research studies 7,8 •viability in cell preparations containing debris DESCRIPTIONFlow cytometry provides a rapid and reliable method to quantify viable cells in eukaryotic and prokaryotic cell suspensions.1-3,7,8 The BD Cell Viability Kit offers an easy-to-use dye combination to distinguish live and dead cells for analysis by flow cytometry. The kit contains thiazole orange (TO) solution to stain all cells and propidium iodide (PI) to stain dead cells. BD Liquid Counting Beads is a liquid suspension of fluorescent beads. Add the beads to a flow sample to calculate absolute counts. The kit can be ordered with or without counting beads. The method provides a rapid alternative to manual microscopic methods.4-6Live cells have intact membranes and are impermeable to dyes such as PI, which leaks into cells with compromised membranes. TO is a permeant dye and enters all cells, live and dead, to varying degrees. The fluorescent signal from TO in viable cells allows their enumeration even when debris in the cell preparation contaminates a scatter gate around the cells. Thus the combination of these two dyes provides a rapid and reliable method for discriminating live and dead eukaryotic and prokaryotic cells, including peripheral blood mononuclear cells (PBMCs), mammalian cell lines, bacteria, and yeast.MATERIALS PROVIDED The kit contains 1 vial of 500 µL 42 µmol/L TO in dimethyl sulfoxide (DMSO) and 1vial of 500 µL 4.3mmol/L PI in water. The optional BD Liquid Counting Beads are supplied as 1 vial of 10 mL of fluorescent microspheres in buffer with 0.1% sodium azide.Material required but not providedRecommended staining buffer: Physiologic phosphate-buffered saline containing 0.2% Pluronic® F68 and 1mmol/L EDTA.23-6755-03Page 2NOTE For bacteria, 0.01% TWEEN® 20 can be substituted for the Pluronic F68. The staining buffer should be passed through a 0.22-µm filter.HANDLING AND STORAGEStore vials at 2°–8°C with the TO and PI stored in the desiccated container provided. Each reagent is stable for the period shown on the bottle label when stored as directed.WARNINGSAll biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 9,10 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves.METHODRecommended amounts of TO and PI depend on the cell type being stained.Dilute cultured cells or PBMCs at least 1:10 in staining buffer to an approximate concentration range of 5x 105 to 107 cells/mL. (See Material required but not provided for preparation method.) For other sample types, such as pharmaceutical, food or environmental samples, at least 100 organisms per mL need to be detected using flow cytometry. If necessary, samples can be brought into this range by an initial concentration step.Bacteria and yeast: Add 5.0 µL of each dye solution to 500 µL of cell suspension. The final staining concentrations are 420 nmol/L for TO and 43 µmol/L for PI. Vortex and incubate for at least 5 minutes at room temperature.Mammalian cells: Add 4.0 µL of TO and 2.0 µL of PI solution to 2 mL of cell suspension. The final staining concentrations are 84 nmol/L for TO and 4.3 µmol/L for PI. Vortex and incubate for 5 minutes at room temperature.Bull semen: Add 2.0 µL of each dye solution to 2 mL of cell suspension. The final staining concentrations are 42 nmol/L for TO and 4.3 µmol/L for PI. Vortex and incubate for 5minutes at room temperature.NOTE When using BD Liquid Counting Beads, allow the beads to come to room temperature. Prior to analysis, gently vortex the bead suspension for 30 seconds and add 50 µL to each tube. Use the reverse pipetting technique to pipette for better accuracy. Cap the tubes and gently vortex to mix. Acquisition and analysis Acquire prepared samples on a BD FACS™ brand flow cytometer equipped with 488-nm laser excitation and BD CellQuest™ software. Use an FSC threshold for mammalian cells (fluorescence threshold if also using BD Liquid Counting Beads), and an SSC threshold for microbial cells. Gate cells using scatter and FL2.4-6 TO fluoresces primarily in FL1 and FL2; PI fluoresces primarily in FL3. Therefore, the best discrimination of live and dead populations is on an FL1 vs FL3 plot.To determine the concentration of the cell populations, use the following equation:Representative dataFigure 1 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a 500-µL sample of E. coli stained with 5 µL of TO and 5 µL of PI. Regions are set around the live, injured, and dead bacterial populations; counting beads are not shown.# events in cell region # events in bead region -----------------------------------------------------------# beads/test*test volume ---------------------------------×dilution factor ×concentration of cell population =* This value is found on the vial of BD Liquid Counting Beads and can vary from lot to lot.Page 323-6755-03Figure 1FL1 vs FL3 dot plot, gated on E. coli by scatter Figure 2 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a 2-mL sample of PBMCs stained with 4 µL of TO and 2 µL of PI. Live cells are in R5; dead cells are in R3; and counting beads are in R4.Figure 2FL1 vs FL3 dot plot, gated on PBMCs by scatter Figure 3 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a 2-mL sample of Raji cells stained with 4 µL of TO and 2 µL of PI. Live cells are in R5; dead cells are in R3; and counting beads are in R4.Figure 3FL1 vs FL3 dot plot, gated on Raji cells by scatter Figure 4 shows results obtained by staining a 2-mL sample of thawed bull semen with 2 µL of TO and 2 µL of PI. For staining, 20 µL of thawed semen was added to 2 mL of staining buffer. Live sperm are in R3, dead sperm are in R2, and injured sperm are between the two regions.R6R5R4live injured dead FL1-H F L 3-H R3R4R5live dead injured FL1-H F L 3-H beads FL1-H F L 3-H R3R4R5dead injured live beads23-6755-03Page 4Figure 4FL1 vs FL3 dot plot, gated on thawed bull semen by scatter LIMITATIONSDispose of stained samples, extra dye solution, and container according to local, state, and federal regulations.CHARACTERIZATION To ensure consistently high-quality reagents, each lot of reagent is tested for conformance with characteristics of a standard reagent. Representative flow cytometric data is included in this data sheet.WARRANTYUnless otherwise indicated in any applicable BD general conditions of sale for non-US customers, the following warranty applies to the purchase of these products.THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER . BD DISCLAIMS HEREBY ALL OTHER WARRANTIES , EXPRESSED OR IMPLIED , INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT . BD ’S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE . BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES , INCLUDING PERSONAL INJURY , OR ECONOMIC LOSS , CAUSED BY THE PRODUCT . REFERENCES1.Nebe-von-Caron G, Stephens PJ, Badley AR. Bacterial detection and differentiation by cytometry and fluorescent probes. Proc Royal Microbiol Society . 1999;34:321-327.2.Nebe-von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA. Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J Microbiol Meth . 2000;42:97-114.3.Davey HM, Kell DB. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev . 1996;60:641-696.4.Alsharif R, Godfrey W . Bacterial detection and live/dead discrimination by flow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002.5.Alsharif R, Tapia M, Godfrey W , Wanalund J, Nagar M. Bacterial disinfectant efficacy using flow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002.6.Thornton R, Godfrey W , Gilmour L, Alsharif R. Evaluation of yeast viability and concentration during wine fermentation using flow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002.7.Graham JK. Assessment of sperm quality: a flow cytometric approach. Anim Reprod Sci . 2001;68:239-247.8.Yamamoto T, Mori S, Yoneyama M, Imanishi M, Takeuchi M. Evaluation of rat sperm by flow cytometry: simultaneous analysis of sperm count and sperm viability. J Toxicol Sci . 1998;23:373-378.9.Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline — Third Edition . Wayne, PA: Clinical and Laboratory Standards Institute; 2005. CLSI document M29-A3.10.Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR . 1988;37:377-388.FL1-H F L 3-H live injured dead R2R3TRADEMARKS AND LICENSE INFORMATION Pluronic is a registered trademark of BASF Corporation.Tween is a registered trademark of Croda International PLCBD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2015 BDThis product is provided under a license agreement between Life Technologies Corporation and Becton, Dickinson and Company through its BD Biosciences business, to use the fluorescent beads in this product only as standards, controls and calibrators for: (a) absolute counting; (b) fluorescence intensity measurements; and (c) instrument setup procedures on flow cytometry instruments, for calibrating and performance testing of flow cytometric instruments and flow cytometric assays, and for internal research and development applications. The sale of this product is expressly conditioned on the buyer not using the fluorescent beads in this product (1) in manufacturing; (2) for therapeutic, diagnostic (except when specifically labeled for such use in connection with BD products), or prophylactic purposes; (3) to resell, even if sold for calibration or research purposes. For information on purchasing a license to this product for purposes other than described above, contact Life Technologies Corporation, Cell Analysis Business Unit, Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300. Fax: (541) 335-0354.Page 523-6755-03。

中国防痨杂志 2021年 4 月第 43卷第 4 期C h i n J A m i t u b e r c，April 2021, Vol. 43，N〇. 4•413••短篇论著•抗结核药品导致严重骨髓抑制一例并文献复习刘鑫郭乐仵倩红【摘要】抗结核药品是引起骨髓抑制的危险因素，严重者可导致患者死亡。

本文作者报道了1例与使用抗结 核药品相关的骨髓抑制并导致死亡的患者。

患者为女性,24岁，以“发现双侧颈部包块1个月余，经外院穿刺病理 诊断为颈部淋巴结结核”住院治疗.入院后颈部包块脓液经结核分枝杆菌药物敏感性试验(M T frD S T)证实为耐多 药结核病，入院常规行血液检查未见异常后给予初治抗结核治疗(3H-R-Z-E/12H-R),全血细胞从最初的仅粒系细 胞轻度降低，逐步发展至重度降低，最终红系、粒系、巨核系细胞均严重降低，从而继发感染性休克;患者被积极抢 救后失败.最终死亡。

作者通过复习相关文献.讨论了抗结核药品引起骨髓抑制的发病机制、临床表现、诊断及治 疗方法，提醒临床医生了解抗结核药品引起的血液系统不良反应及其严重性，尽早明确原因并采取必要的干预措施。

【关键词】抗结核药；结核，抗多种药物性；骨髓疾病；死亡；综述文献(主题）A case of severe myelosuppression caused by anti-tuberculosis drugs and literature review on this issue LIU X i n,GUO L e*WJJ Q ian-hong. Shaanxi Provincial Tuberculosis Prevention and Control Institute* X i1 an 710100 <,ChinaCorresponding author：W U Qian-hong, Email ：15902969^31 @126. com【Abstract】A nti-tuberculosis drug is a risk factor for causing bone m arrow suppression，which can lead to death in vSevere cases. T his article reported a patient who got bone m arrow suppression caused by the use of anti­tuberculosis drugs and died. T he authors also did a literature review regarding to this issue. T he patient was female, 24 years old. She was found to have bilateral neck masses for m ore than 1m onth and diagnosed as cervical lymph node tuberculosis by puncture biopsy in another hospital before she came to our hospital. A fter adm ission,she was confirmed as m ultidrug-resistant tuberculosis (M DR-TB) by Mycobacterium tuberculosis drug susceptible test (M T&-DST) using the pus obtained from her neck mass. T he routine blood examinations were performed for her and the results w ere norm al, and then the initial treatm ent regimen of anti-tuberculosis (3H-R-Z-E/12H-R) was given to this patient. A fter she received the treatm ent, the granulocyte cells were starting to drop slightly and gradually progressed to severe reduction，and then further progressed to severe reductions of the erythroid，granulocyte and m egakaryocyte cells. T he patient eventually died of infectious shock and failure to resuscitate. By reviewing the relevant publications, the authors did discussions on pathogenesis, clinical m anifestations, diagnosis and treatm ent of bone m arrow suppression caused by anti-tuberculosis drugs in this article, aimed to remind clinicians to understand the adverse reactions in blood system caused by anti-tuberculosis drugs and their severity,and to emphasize that the causes of the abnormal blood test results should be identified as soon as possible and the effective intervention m easures should be taken soon.【Key words】A ntitubercular agents; Tuberculosis，m uhidrug-resistant; Bone m arrow diseases；D eath；Review literature as topic骨髓抑制是抗结核药品治疗中经常发生的且严重的药 物不良反应(adverse drug reaction，A D R)，相关文献报道发 生率在3. 3%〜10%[11。