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首页> 外文期刊>Journal of Immunological Methods >A flow cytometric method to assess antigen-specific proliferative responses of different subpopulations of fresh and cryopreserved human peripheral blood mononuclear cells.
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A flow cytometric method to assess antigen-specific proliferative responses of different subpopulations of fresh and cryopreserved human peripheral blood mononuclear cells.

机译：流式细胞术评估新鲜和冷冻保存的人外周血单个核细胞不同亚群的抗原特异性增殖反应。

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摘要

We have used PKH26 dye, which is incorporated stably into the membrane of cells, to determine, using flow cytometry, lymphocyte proliferative responses to the antigen tetanus toxoid in fresh and cryopreserved samples. Measuring cell proliferation with this dye has advantages over either 3H-thymidine or Bromodeoxyuridine (BrdU). Whereas the existing methods measure proliferation at a single time point, PKH26 gives a cumulative measure of cell proliferation. As PKH26 is incorporated into the cell membrane, cells do not have to be permeabilised to allow dye incorporation into a cytoplasmic compartment. Most importantly, PKH26 can be used in combination with monoclonal antibodies to surface markers on mixed populations of cells, to determine the proliferation of individual subpopulations, without the need for prior cell fractionation. We also show that PKH26 can be used with similar efficacy in both fresh and cryopreserved samples. In addition since PKH26 is a cumulative measure of proliferative responses we were able to show that restimulation of the dividing population in vitro with fresh antigen presenting cells (APC) and antigen permits characterisation of a further proliferating cell population. The use of PKH26 dye in combination with cell phenotyping and measurement of cytokine production at the single cell level will prove a powerful tool for multiparameter analyses of cellular responses to antigen.
机译：我们已经使用了PKH26染料，该染料被稳定地掺入细胞膜中，使用流式细胞仪来确定新鲜和冷冻保存的样品中对破伤风类毒素类毒素的淋巴细胞增殖反应。与3H-胸苷或溴脱氧尿苷（BrdU）相比，用这种染料测量细胞增殖具有优势。现有方法在单个时间点测量增殖，而PKH26则是细胞增殖的累积测量。由于PKH26被掺入细胞膜中，因此不必渗透细胞即可使染料掺入细胞质区室。最重要的是，PKH26可以与抗混合细胞群体表面标志物的单克隆抗体结合使用，从而确定单个亚群的增殖，而无需事先进行细胞分级分离。我们还表明，PKH26可以在新鲜和冷冻样品中以相似的功效使用。此外，由于PKH26是增殖反应的累积量度，因此我们能够证明，使用新鲜的抗原呈递细胞（APC）和抗原在体外对分裂群体进行再刺激可以表征进一步增殖的细胞群体。 PKH26染料与细胞表型的结合使用以及在单细胞水平上测量细胞因子的产生将被证明是对抗原对细胞反应进行多参数分析的强大工具。

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