Cory J M, Rapp F, Ohlsson-Wilhelm B M
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey 17033.
Cytometry. 1990;11(5):647-51. doi: 10.1002/cyto.990110514.
Effects of cell fixation procedures appropriate for flow cytometric analysis on the infectivity of human T lymphoblastoid H9 cells infected with human immunodeficiency virus-1 (HIV-1) were evaluated to provide guidelines for choosing cell treatments for potentially infectious samples. H9 cells experimentally infected with HIV-1 were treated by the test fixation procedure, washed, and cocultured with equal numbers of live, uninfected H9 cells. To estimate the reduction in infectivity due to the fixation procedure, dilution series of live infected H9 cells in uninfected H9 cells were simultaneously established in culture. Cell cultures were incubated 8-10 d, harvested, and evaluated for evidence of HIV-1 infection by the presence of cell-associated HIV-1 antigens and/or by the presence of particle-associated reverse transcriptase activity in cell culture supernatants. Thirty-minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehyde (0.5%) reduced the infectivity of HIV-1-infected H9 cells by greater than 99.99%. To the same degree, a multi-step fixation procedure utilizing formaldehyde and ethanol was effective in reducing HIV-1 infectivity. Conversely, the erythrocyte fixative dimethylsuberimidate at 3 micrograms/ml was ineffective in reducing HIV-1 infectivity.
评估了适用于流式细胞术分析的细胞固定程序对感染人类免疫缺陷病毒1型（HIV-1）的人T淋巴母细胞H9细胞感染性的影响，以提供针对潜在感染性样本选择细胞处理方法的指导原则。用测试固定程序处理经实验感染HIV-1的H9细胞，洗涤后，与等量的未感染活H9细胞共培养。为了估计固定程序导致的感染性降低，在培养中同时建立未感染H9细胞中活感染H9细胞的稀释系列。细胞培养物孵育8 - 10天，收获后，通过细胞相关HIV-1抗原的存在和/或细胞培养上清液中颗粒相关逆转录酶活性的存在来评估HIV-1感染的证据。用1.85%甲醛、无水甲醇、甲醇：丙酮（1:1）或0.5%多聚甲醛固定30分钟可使感染HIV-1的H9细胞的感染性降低超过99.99%。同样程度地，利用甲醛和乙醇的多步固定程序在降低HIV-1感染性方面有效。相反，3微克/毫升的红细胞固定剂亚胺二甲酯在降低HIV-1感染性方面无效。
