Schimenti K J, Jacobberger J W
Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106.
Cytometry. 1992;13(1):48-59. doi: 10.1002/cyto.990130109.
Mammalian tissue culture cells were fixed with 3 different alcoholic fixatives--acetone:methanol, EtOH, and MeOH. The quality of the resulting DNA histograms was evaluated by comparison of CV, G1/G2 ratio, G1 mode, cell aggregation, and debris formation; 81-90% MeOH (final concentration) was determined to be the optimal fixative by these criteria. A procedure was then examined using a prefix with paraformaldehyde followed by MeOH (PF/MeOH). This procedure produced cell preparations with reduced debris and aggregation, equivalent mode and ratio, but increased CV when compared with MeOH fixation. Both MeOH and PF/MeOH fixation procedures were then compared for their utility in dual staining for DNA and intracellular immunofluorescence for a nuclear protein, SV40 T antigen (Tag). Since alcohols are known to affect immunofluorescence staining of some antigens, fixation with paraformaldehyde followed by Triton X-100 permeabilization (PF/TX) was also included in this comparison to generalize the study by providing an alternative to MeOH permeabilization. The three procedures were evaluated for the quality of the sample by measuring the same descriptors of the DNA parameter as in the alcohol study. PF/TX consistently produced samples with decreased DNA CV and less debris and aggregation compared to MeOH methods. Two criteria were used to evaluate immunofluorescence--the amount of Tag measured and reproducibility. All MeOH methods were equivalently reproducible with CV's less than 3%. PF/TX was slightly less so with a CV of less than 6%. In contrast, different levels of Tag were measured for each procedure. For mouse 3T3 cells infected with a recombinant retroviral vector encoding T antigen, the level of T antigen measured after PF/MeOH was 21% greater than in MeOH fixed cells, and the level in PF/TX fixed cells was 37% less. The fraction of fluorescence specific to T antigen for these cells was 79-83% for all procedures. The lower levels measured after fixation by PF/TX were shown to be due to epitope masking. Why higher levels are measured with PF/MeOH procedures is unknown at present but may be due to antigen retention. Therefore, each of these fixation methods may be used with confidence in reliability but they are not equivalent with respect to the molecular architecture of the nucleus. It is postulated that PF/TX permeabilizes cells but cells retain native supramolecular structure, whereas MeOH based fixatives disrupt this structure and randomize availability of epitope to antibody. If so, the two procedures could be used as complementary procedures to study gene expression and function.
哺乳动物组织培养细胞用3种不同的酒精固定剂——丙酮:甲醇、乙醇和甲醇进行固定。通过比较变异系数（CV）、G1/G2比值、G1峰、细胞聚集和碎片形成情况来评估所得DNA直方图的质量；根据这些标准，确定81 - 90%甲醇（终浓度）为最佳固定剂。然后研究了一种先用多聚甲醛预处理再用甲醇处理（PF/MeOH）的方法。与甲醇固定相比，该方法制备的细胞样本碎片和聚集减少，峰和比值相当，但CV增加。然后比较了甲醇和PF/MeOH固定方法在DNA双染和核蛋白SV40 T抗原（Tag）细胞内免疫荧光检测中的效用。由于已知酒精会影响某些抗原的免疫荧光染色，因此在该比较中还包括先用多聚甲醛固定再用Triton X - 100通透处理（PF/TX）的方法，以便通过提供甲醇通透处理的替代方法来推广该研究。通过测量与酒精研究中相同的DNA参数描述符来评估这三种方法的样本质量。与甲醇方法相比，PF/TX始终能产生DNA变异系数降低、碎片和聚集较少的样本。使用两个标准来评估免疫荧光——测量的Tag量和可重复性。所有甲醇方法的可重复性相当，变异系数小于3%。PF/TX的可重复性略低，变异系数小于6%。相比之下，每种方法测量的Tag水平不同。对于感染了编码T抗原的重组逆转录病毒载体的小鼠3T3细胞，PF/MeOH处理后测量的T抗原水平比甲醇固定的细胞高21%，而PF/TX固定的细胞中的水平低37%。所有方法中这些细胞针对T抗原的荧光比例为79 - 83%。结果表明，PF/TX固定后测量的较低水平是由于表位掩盖。目前尚不清楚PF/MeOH方法测量的较高水平的原因，但可能是由于抗原保留。因此，这些固定方法在可靠性方面都可以放心使用，但就细胞核的分子结构而言它们并不等效。据推测，PF/TX使细胞通透，但细胞保留天然超分子结构，而基于甲醇的固定剂会破坏这种结构并使表位与抗体的结合随机化。如果是这样，这两种方法可以作为互补方法用于研究基因表达和功能。
