实验方案
简要概述
- 准备实验细胞
- 溶液配制
- 在室温或37℃下将染料与细胞一起孵育15至30分钟
- 去除染料中的工作溶液
- 使用流式细胞仪分析（Ex / Em = 490/520 nm-FITC滤波器）
溶液配制
储备溶液配制
CytoTrace Ultra Green原液（2-10 mM）：添加适量的DMSO充分混合以制成CytoTrace Ultra Green原液（2-10 mM）。注意：储备溶液应立即使用；所有剩余的溶液应等分，并在<-20 o C下冷冻。避免重复冻融循环，并避光。
工作溶液配制
CytoTrace Ultra Green工作溶液：通过用Hanks和20 mM Hepes缓冲液（HHBS）或自备的pH=7的缓冲液稀释DMSO储备液，制备0.5至5 µM的染料工作液，并离心。注意：在某些细胞类型，可能需要较低的浓度才能染色细胞。我们建议在进行实验之前针对每种细胞类型进行测试以获得佳浓度使实验更顺利。
操作步骤
- 用测试化合物处理细胞。
- 离心细胞以获得每管2-10×10 5个细胞。
- 将细胞重悬于500 µL CytoTrace Ultra Green工作溶液中。
- 在室温或37°C下，将细胞与染料溶液孵育15至30分钟，避光。
- 从细胞中除去染料工作溶液；可选：4％甲醛固定细胞。
- 用HHBS或自备的缓冲液洗涤细胞一次。
- 将细胞重悬于500 µL预热的HHBS或培养基中，每管可得到2-10×10 5个细胞。
- 使用流式细胞仪或带有FITC滤波片组的荧光显微镜检测Ex / Em = 490/520 nm处的荧光变化。
图示
图1. Jurkat细胞中CytoTrace Ultra Green与CMFDA的荧光强度比较。在37 ℃，5％CO2培养箱中，将Jurkat细胞用CytoTrace Ultra Green和CMFDA染料分别加载30分钟。使用带有FITC通道的ACEA NovoCyte 3000流式细胞仪测量荧光强度。
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相关产品
| 货号 # |
产品名称 |
规格 |
分子量 |
Ex/Em (nm) |
溶剂 |
| 22014 |
CytoTrace Orange CMTMR |
10×50 mg |
554.04 |
541/565 |
DMSO |
| 22015 |
CytoTrace Red CMPTX |
10×50 mg |
686.25 |
577/602 |
DMSO |
| 22016 |
CytoTrace Red CFDA |
1 mg |
652.43 |
560/574 |
DMSO |
| 22017 |
CytoTrace Green CMFDA |
1 mg |
464.86 |
494/521 |
DMSO |
| 21800 |
CytoTrace Ultra Green |
1 mg |
822.72 |
494/521 |
DMSO |
| 22020 |
FDA (Fluorescein diacetate) |
1 g |
416.83 |
494/521 |
DMSO |
