Use of DiO-C5-3 to improve Hoechst 33342 uptake, resolution of DNA content, and survival of CHO cells.
Use of DiO-C5-3 to improve Hoechst 33342 uptake, resolution of DNA content, and survival of CHO cells.
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使用 DiO-C5-3 提高 Hoechst 33342 的摄取、DNA 含量的分辨率以及 CHO 细胞的存活率。
DOI：
10.1016/0014-4827(88)90309-6
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发表时间：
1988
影响因子：
3.7
通讯作者：
Tobey,RA
中科院分区：
文献类型：
--
作者：
Crissman,HA;Hofland,MH;Stevenson,AP;Wilder,ME;Tobey,RA
中国仓鼠细胞系CHO用9μMHoechst 33342(HO)单独或与膜电位荧光染料Dio-C5-3(DIO)联合染色，用25~500 mW的紫外激光功率进行分析，并通过集落形成实验测定存活率。在细胞活力不受影响的条件下，HO-Dio的组合使荧光增加了两倍，变异系数(CVs)低至3.0%，即使在500 mW的激发下也不受影响。在类似的分析条件下，HO染色的细胞产生约8.5%的CVS和约90%的存活率。在25 mW的激光功率下，HO-Dio染色群体的CV值为4.0%，而HO-Dio染色细胞的CV值为9.4%。结果用另一种膜电位染料罗丹明123(R123)与HO联合使用时，与HO染色的细胞相比无明显改善。在HO或HO-Dio染色的人群中，没有观察到优先的、细胞周期特定的杀伤作用。在整个激光功率范围内，HO或HO-Dio染色的人皮肤二倍体成纤维细胞的CVS是相似的；然而，当在更高的功率(400-500 mW)范围内分析和分类时，HO-Dio染色的人群的存活率略高。用HO-Dio染色的细胞建立了DNA倍性不同的分选CHO-K1亚群的长期培养。这种新染色方法的优点包括提高了DNA含量的分辨率(低CV值)，以及可能使用更便宜的FCM UV激光系统，以及更少的扰动激发功率。
Chinese hamster cells (line CHO) stained with either 9 μMHoechst 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 (R123), 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.