Crissman H A, Hofland M H, Stevenson A P, Wilder M E, Tobey R A
Los Alamos National Laboratory, National Flow Cytometry Resource, New Mexico 87545.
Exp Cell Res. 1988 Feb;174(2):388-96. doi: 10.1016/0014-4827(88)90309-6.
Chinese hamster cells (line CHO) stained with either 9 microM Hoechst 33342 (HO) alone or in combination with the membrane potential fluorochrome DiO-C5-3 (DiO) were analyzed using uv laser powers between 25 and 500 mW and sorted for determination of survival by a colony formation assay. The combination of HO-DiO increased fluorescence twofold and provided coefficients of variation (CVs) as low as 3.0% under conditions where viability of cells, even at 500 mW excitation, was unaffected. HO-stained cells yielded CVs of about 8.5% and survivals of approximately 90% under similar analytical conditions. At laser powers of 25 mW, CV values for HO-DiO-stained populations were 4.0% compared to 9.4% for HO-stained cells. Results with another membrane potential dye, rhodamine 123 (R 123), in combination with HO showed no improvement compared to HO-stained cells. No preferential, cell cycle phase-specific killing was observed in either the HO- or HO-DiO-stained populations. CVs of human skin diploid fibroblasts stained with HO or with HO-DiO were comparable over the entire laser power range; however, percentage survival was slightly higher for the HO-DiO-stained populations when analyzed and sorted at the higher power (400-500 mW) range. Long-term cultures of sorted CHO-K1 subpopulations, differing in DNA ploidy, were established from HO-DiO-stained cells. Advantages of this new staining procedure include improved DNA content resolution (low CV values) and the potential use of less expensive FCM uv laser systems coupled with less perturbing excitation powers.
使用25至500毫瓦的紫外激光功率对单独用9微摩尔的Hoechst 33342（HO）或与膜电位荧光染料DiO-C5-3（DiO）联合染色的中国仓鼠细胞（CHO细胞系）进行分析，并通过集落形成试验分选以确定存活率。HO-DiO组合使荧光增强了两倍，并且在细胞活力不受影响的条件下（即使在500毫瓦激发下）提供了低至3.0%的变异系数（CV）。在类似的分析条件下，HO染色的细胞产生约8.5%的CV和大约90%的存活率。在25毫瓦的激光功率下，HO-DiO染色群体的CV值为4.0%，而HO染色细胞的CV值为9.4%。另一种膜电位染料罗丹明123（R 123）与HO联合使用的结果与HO染色细胞相比没有改善。在HO或HO-DiO染色群体中均未观察到优先的、细胞周期阶段特异性杀伤。用HO或HO-DiO染色的人皮肤二倍体成纤维细胞的CV在整个激光功率范围内相当；然而，当在较高功率（400 - 500毫瓦）范围内分析和分选时，HO-DiO染色群体的存活百分比略高。从HO-DiO染色细胞建立了DNA倍性不同的分选CHO-K1亚群的长期培养物。这种新的染色方法的优点包括提高了DNA含量分辨率（低CV值）以及有可能使用成本较低的流式细胞术紫外激光系统和干扰较小的激发功率。
