Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells.
Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells.
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DOI：
10.4049/jimmunol.136.8.2769
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发表时间：
1986-04
影响因子：
4.4
通讯作者：
P. Rabinovitch;R. Torres;D. Engel
中科院分区：
文献类型：
--
作者：
P. Rabinovitch;R. Torres;D. Engel
DNA结合的荧光染料7-氨基-放线菌素D（7AAD）可被流式细胞术中常用的488 nm激光线有效激发，但产生的荧光发射比其他DNA特异性荧光染料更远。在这份报告中，我们表明，7AAD的光谱特性允许单激光分析的DNA含量和细胞周期，同时与两个细胞表面标记物标记荧光素（绿色）和藻红蛋白（橙子）结合的抗体。7AAD的使用使得三色分析实用可行，使用最广泛可用的流式细胞仪。这种技术的力量在两个系统中得到了证明。染色的人外周血淋巴细胞（PBL）与7AAD被证明是依赖于细胞活化和染色质构象; PHA刺激的细胞已成为活化和表达IL 2受体有更大的7AAD荧光比非活化，IL 2受体阴性细胞。用荧光抗体IgM和Ly-1染色的小鼠脾细胞的细胞周期分析表明，天然脾中S和G2期细胞的比例在用这些标记物鉴定的细胞亚群之间变化很大。特别令人感兴趣的是令人震惊的发现，即Ly-1+/IgM+亚群（Ly-1 B细胞）极大地富集了S期部分中的细胞。这是重要的，因为Ly-1 B细胞与自身抗体的产生有关，并且与这些细胞具有淋巴母细胞样或浆母细胞形态的报道一致。我们推测Ly-1 B细胞可能属于体内活化细胞的一个亚群，这些细胞要么快速增殖，要么被阻滞在S期。
The DNA-binding, fluorescent dye 7-amino-actinomycin D (7AAD) is efficiently excited by the 488 nm laser line commonly used in flow cytometry, but yields fluorescence emission further into the red spectrum than alternative DNA-specific fluorochromes. In this report, we show that the spectral properties of 7AAD allow single-laser analysis of DNA content and cell cycle simultaneously with two cell surface markers labeled with fluorescein (green)-and phycoerythrin (orange)-conjugated antibodies. The use of 7AAD makes three-color analysis practical and feasible, using the most widely available flow cytometric instruments. The power of this technique was demonstrated in two systems. Staining of human peripheral blood lymphocytes (PBL) with 7AAD was demonstrated to be dependent on cell activation and chromatin conformation; PHA-stimulated cells which have become activated and express IL 2 receptors had greater 7AAD fluorescence than nonactivated, IL 2 receptor-negative cells. Cell cycle analysis of mouse splenocytes stained with fluorescent antibodies to IgM and to Ly-1 demonstrated that the proportion of S and G2 phase cells in native spleen varies strongly among the subsets of cells identified with these markers. Of particular interest was the striking finding that the Ly-1+/IgM+ subset (Ly-1 B cells) is greatly enriched for cells in the S phase fraction. This is important because Ly-1 B cells have been associated with the production of autoantibodies, and is consistent with reports that these cells have a lymphoblastoid or a plasmablast morphology. We hypothesize that Ly-1 B cells may belong to a subset of in vivo activated cells which are either rapidly proliferating or are arrested in S phase.