Frey T
Becton Dickinson Immunocytometry Systems, San Jose, California 95131, USA.
Cytometry. 1995 Nov 1;21(3):265-74. doi: 10.1002/cyto.990210307.
Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub-G1 DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy-1 dim thymocytes were included in the dyc dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL-60 cells treated with camptothecin. However, the dyes SYTO-16 and LDS-751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon-ion laser.
当用几种核酸染料染色时，发现凋亡胸腺细胞比正常胸腺细胞暗淡得多。这些染料为凋亡提供了一种快速简便的检测方法，适用于活细胞，且不需要紫外激光。呈现这种染色模式的染料集合包括适用于标准FACScan的FL1、FL2或FL3通道的试剂。用这些试剂鉴定的细胞与根据几种广泛使用的标准定义的凋亡胸腺细胞相同：（i）快速摄取Hoechst 33342但排斥碘化丙啶，（ii）部花青540明亮，以及（iii）在能洗脱片段化DNA的缓冲液中通透后DNA含量低于G1期。此外，L3T4/Thy-1暗淡的胸腺细胞也包含在染料暗淡的群体中。在用喜树碱处理的HL-60细胞中，标准的Hoechst 33342和部花青540检测方法无法区分正常细胞和凋亡细胞群体。然而，在这个模型系统中，染料SYTO-16和LDS-751都能充分区分凋亡细胞和非凋亡细胞。其中一些染料在流式细胞仪的其他荧光通道中发出的信号也非常少，可用于仅配备单个氩离子激光的细胞仪上的多色检测。
