Lecoeur H, Gougeon M L
Département SIDA et Rétrovirus, Institut Pasteur, Paris, France.
J Immunol Methods. 1996 Oct 30;198(1):87-99. doi: 10.1016/0022-1759(96)00148-2.
The present article reports a multiparametric cytofluorimetric analysis of apoptosis in murine thymocytes and human PBMC from healthy donors or HIV-infected patients. We have evaluated four previously described cytofluorimetric methods of apoptosis quantification, each of them detecting distinct cellular alterations of the apoptosis process. Reduced DNA stainability was detected with the PI assay on nuclei and the AO/EB dual staining method was evaluated on entire and non-fixed cells. DNA strand breaks were detected following in situ nick translation, and alterations in membrane integrity were evaluated following 7-AAD incorporation. When apoptosis was quantified in murine thymocytes under various conditions of induction, the combined analysis of FSC/SSC criteria and 7-AAD or AO/EB staining on the same samples permitted the identification of distinct steps in the apoptosis process. Moreover these four methods proved to be reliable and gave statistically similar results both on murine thymocytes and PBMC from healthy donors. However, in HIV-infected persons, some discordant apoptosis determinations were observed with PI and 7-AAD staining assays. We found that after Ficoll isolation, PBMC from AIDS patients were enriched in erythrocytes and granulocytes. On the one hand, granulocytes were found to be responsible for a poor apoptosis estimation with the PI assay whereas erythrocytes were responsible for an underestimation rate of apoptosis in the 7-AAD assay. To prevent such interference, we propose some modifications which render these methods more suitable for application to PBMC from HIV-infected patients. Taken together these observations indicate that it is essential to assess critically the apoptosis quantification methods with respect to their applicability to complex lymphoid populations such as those from AIDS patients.
本文报道了对来自健康供体或HIV感染患者的小鼠胸腺细胞和人外周血单核细胞（PBMC）凋亡的多参数细胞荧光分析。我们评估了四种先前描述的细胞荧光凋亡定量方法，每种方法检测凋亡过程中不同的细胞变化。用PI检测细胞核来检测DNA染色性降低，并用AO/EB双重染色法对完整的非固定细胞进行评估。原位缺口平移后检测DNA链断裂，7-AAD掺入后评估膜完整性变化。在各种诱导条件下对小鼠胸腺细胞凋亡进行定量时，对同一样本的FSC/SSC标准与7-AAD或AO/EB染色进行联合分析，可确定凋亡过程中的不同阶段。此外，这四种方法被证明是可靠的，并且在小鼠胸腺细胞和健康供体的PBMC上给出的统计结果相似。然而，在HIV感染者中，PI和7-AAD染色检测观察到一些不一致的凋亡测定结果。我们发现，Ficoll分离后，艾滋病患者的PBMC富含红细胞和粒细胞。一方面，发现粒细胞导致PI检测凋亡估计不佳，而红细胞导致7-AAD检测凋亡低估率。为防止这种干扰，我们提出了一些改进措施，使这些方法更适用于HIV感染患者的PBMC。综上所述，这些观察结果表明，至关重要的是要严格评估凋亡定量方法对复杂淋巴细胞群体（如艾滋病患者的淋巴细胞群体）的适用性。
