上海交通大学学报(医学版)

• 技术与方法 • 上一篇    下一篇

采用EDTA-胰酶处理难消化细胞株在凋亡检测分析中的可行性

符蓉,赵犇鹏,杨洁,田烨   

  1. 上海交通大学 基础医学院生物化学与分子细胞生物学系公共平台流式实验室, 上海 200025
  • 出版日期:2015-09-28 发布日期:2015-09-30
  • 通讯作者: 田烨, 电子信箱: alpha1217@163.com。
  • 作者简介:符蓉(1987—), 女, 助理实验师, 学士; 电子信箱: furong@shsmu.edu.cn。

Feasibility on treating indigestible cell lines by trypsin-EDTA solution for detection of cell apoptosis

FU Rong, ZHAO Ben-peng, YANG Jie, TIAN Ye   

  1. Flow Cytometry Facility, Department of Biochemistry and Molecular Cell Biology, Basic Medical College, Shanghai Jiao Tong University, Shanghai 200025, China
  • Online:2015-09-28 Published:2015-09-30

摘要:

目的  研究细胞胰酶消化液中有无EDTA对流式细胞术检测细胞凋亡率的影响。方法  以7种难消化细胞株作为实验对象,分为对照组和H2O2组(加H2O2促凋亡)。采用不同方法(不含EDTA的0.25%胰酶、含EDTA的0.25%胰酶、含EDTA的0.25%胰酶消化离心弃上清之后用1×PBS洗1次)对细胞进行消化处理,利用Annexin V-FITC荧光抗体标记细胞,流式细胞术检测细胞凋亡率。计算相同消化细胞条件下两组细胞的凋亡率差值。结果  统计学分析结果显示:7种细胞株在3种消化细胞条件下的细胞凋亡率差值比较,差异均无统计学意义(P>0.05)。结论  胰酶中含EDTA对流式细胞术检测细胞凋亡无显著影响;因此可以利用EDTA抗凝集这一特性,将其加入胰酶消化液中,用于某些难消化细胞株凋亡样品的制备。

关键词: 流式细胞术, EDTA, 凋亡, Annexin V, 胰酶

Abstract:

Objective  To explore the effect of the presence of EDTA in trypsin solution on detecting the apoptosis rate by flow cytometry method (FCM). Methods  Seven indigestible cell lines were divided into the control group and experimental group (treated by H2O2 to promote apoptosis). Cells were treated by different methods (digested by 0.25% trypsin without EDTA, 0.25% trypsin with EDTA, and 0.25% trypsin with EDTA and washed by 1×PBS after being centrifugated and supernatant was removed). Cells were labeled by Annexin V-FITC fluorescent antibodies and the apoptosis rate was detected by flow cytometry (FCM). The difference of apoptosis rate under the same digestion condition between two groups was calculated. Results  Results of statistical analysis showed that differences of the apoptosis rate of seven cell lines under three digestion conditions were not statistically significant (P>0.05). Conclusion  The presence of EDTA in trypsin solution has no significant effect on detecting the cell apoptosis by FCM. Given the anti-agglutinating characteristic of EDTA, it can be added into the trypsin solution for preparation of apoptosis samples for some indigestible cell lines.

Key words: flow cytometry method, EDTA, apoptosis, Annexin V, trypsin