Diaz-Romero Jose, Vogt Gerard, Weckbecker Gisbert
Department of Transplantation, Novartis Pharma AG, Basel, Switzerland.
Cytometry. 2002 Apr 1;47(4):265-75. doi: 10.1002/cyto.10081.
Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood.
In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3).
After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples).
We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.
通过流式细胞术检测细胞内分子时，细胞通透化通常与全血不相容。本文描述了一种同时检测大鼠全血中表面抗原和DNA含量的新技术。
在20微升大鼠全血中，使用标准红细胞裂解试剂（Erythrolyse）使细胞通透化以进行DNA染色。通过使用三种表面标志物（CD3、CD4和CD8α）和一种DNA结合染料（TO-PRO-3）的组合，通过流式细胞术实现免疫表型分析和细胞凋亡检测。
全血与1微摩尔地塞米松孵育24小时后，凋亡淋巴细胞通过其减小的大小和DNA含量与正常淋巴细胞明显区分开来。地塞米松诱导的凋亡细胞百分比，CD4 + T细胞为58.9±4.6，CD8 + T细胞为77.4±2.9，而在无地塞米松时，CD4 + T细胞为12.6±2.7，CD8 + T细胞为17.2±3.5（来自10只动物的重复样本数据）。
我们开发了一种使大鼠全血微量样本中有核细胞通透化的新技术。该方法允许在大鼠全血中同时进行免疫表型分析和细胞凋亡检测。
