多聚甲醛
碘化丙啶
免疫荧光
染色
流式细胞术
固定(群体遗传学)
生物
细胞内
分子生物学
化学
生物物理学
生物化学
抗体
免疫学
细胞凋亡
有机化学
基因
遗传学
程序性细胞死亡
作者
Agnese A. Pollice,J. Philip McCoy,Stanley E. Shackney,Charles A. Smith,Jyotsna Agarwal,Dennis R. Burnolt,Laura Janocko,Francis J. Hornicek,Sarita Singh,Robert J. Hartsock
出处
期刊:Cytometry
[Wiley]
日期:1992-01-01
卷期号:13 (4): 432-444
被引量:111
标识
DOI:10.1002/cyto.990130414
摘要
Abstract A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol‐fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone ( p < 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde ( p < 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti‐leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity oaf DNA staining with propidium iodide were dependent on paraformaldehyde concentration and Fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37°C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation‐induced effect is useful in identifying biologically distinct near‐diploid subpopulations in tumors.
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