样品实验方案
简要概述
1.用测试化合物准备细胞
2.加入1X染料工作液
3.在室温或37oC下将染料与细胞一起孵育10至30分钟
4.去除染料工作液
5.使用流式细胞仪和适当的滤波片进行分析
溶液配制
1.储备溶液配制
所有未使用的储备溶液应分为一次性使用的等分试样,并在制备后储存在-20°C下。避免重复冻融循环。
CytoTell 染料储备液(500X):
将500 µL DMSO加入到染料粉瓶中,通过涡旋使其充分混合,得到储备液(500X)。注意:储备溶液应及时使用;应将所有剩余的溶液等分并在<-20 o C下冷冻。避免重复冻融循环,并避光。
2.工作溶液配制
CytoTell 染料工作溶液(1X):
在Hanks和20 mM Hepes缓冲液(HHBS)或您选择的pH 7缓冲液中以1至500稀释500X DMSO储备溶液(例如1到500的DMX储备溶液稀释到500 µL缓冲液)。涡旋混合均匀。注意:对于不同的细胞类型和/或实验条件,应根据经验确定染料工作溶液的最终浓度。建议以至少十倍以上的浓度进行测试。例如,CytoTell Red在某些细胞类型中使用的量可能要比建议的浓度少得多。
样品示例及操作
1.用测试化合物处理细胞所需的时间。
2.离心细胞以获得每管1-5×105个细胞。
3.在500 µL CytoTell 染料工作溶液中重悬细胞。可选:可以将500X DMSO储备溶液直接添加到细胞中而无需去除培养基(例如,将500 µL DMSO储备溶液添加到500 µL细胞中)
4.在室温或37°C下,将细胞与染料溶液孵育10至30分钟,避光。
5.从细胞中除去染料工作溶液,用HHBS或您选择的缓冲液洗涤细胞。将细胞重悬于500 µL预热的HHBS或培养基中,每管可得到1-5×10 5个细胞。
6.用流式细胞仪或荧光显微镜监测在Ex / Em(参见表1)下的荧光变化。
参考文献
Cardiomyocyte cell cycle dynamics and proliferation revealed through cardiac-specific transgenesis of fluorescent ubiquitinated cell cycle indicator (FUCCI)
Authors: R. Alvarez
Journal: J Mol Cell Cardiol (2019): 154-164
In Vivo Fluorescence Microendoscopic Monitoring of Stent-Induced Fibroblast Cell Proliferation in an Esophageal Mouse Model
Authors: E. J. Jun
Journal: J Vasc Interv Radiol (2018): 1756-1763
Visualizing Breast Cancer Cell Proliferation and Invasion for Assessing Drug Efficacy with a Fluorescent Nanoprobe
Authors: M. Luan
Journal: Anal Chem (2017): 10601-10607
A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution
Authors: J. Begum
Journal: Cytometry A (2013): 1085-95
Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice
Authors: A. M. Vacaru
Journal: Genesis (2013): 751-62
Monitoring cell proliferation in vitro with different cellular fluorescent dyes
Authors: J. Zolnierowicz
Journal: Folia Histochem Cytobiol (2013): 193-200
Long-term monitoring of live cell proliferation in presence of PVP-Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE
Authors: R. Penjweini
Journal: J Microsc (2012): 100-8
Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation
Authors: H. He
Journal: Luminescence (2012): 310-4
Assessment of cell proliferation with resazurin-based fluorescent dye
Authors: E. M. Czekanska
Journal: Methods Mol Biol (2011): 27-32
Study on effect of peptide-conjugated near-infrared fluorescent quantum dots on the clone formation, proliferation, apoptosis, and tumorigenicity ability of human buccal squamous cell carcinoma cell line BcaCD885
Authors: D. Sun
Journal: Int J Nanomedicine (2010): 401-5
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