Popma S H, Krasinskas A M, McLean A D, Szeto W Y, Kreisel D, Moore J S, Rosengard B R
Department of Surgical Research, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Cytometry. 2000 Oct 15;42(5):277-83. doi: 10.1002/1097-0320(20001015)42:5<277::aid-cyto4>3.0.co;2-a.
Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient.
异种移植需要在体外监测复杂的细胞相互作用。一种能够详细监测细胞增殖的工具，对于理解异种淋巴细胞混合培养物以及异种移植患者体内的这些细胞相互作用将具有重要作用。为实现这一目标，我们将荧光细胞增殖标记物5,6 - 羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）与流式细胞术结合使用。CFSE是一种绿色荧光分子，可与细胞内大分子共价结合。每次细胞分裂会使每个细胞的荧光强度减半，并在流式细胞术分析中呈现出特征性的多峰模式。在本研究中，人类淋巴细胞用CFSE标记，并在经辐照的猪淋巴细胞存在的情况下进行培养。在单参数和多参数流式细胞术设置中均检测到CFSE标记的淋巴细胞中的细胞增殖。同时，评估了常用的测量总体细胞增殖的方法——氚化（³H）胸腺嘧啶核苷掺入。CFSE标记的细胞增殖动力学与³H - 胸腺嘧啶核苷掺入相关，因为两种方法在第1 - 3天均显示出延迟期，在第4 - 7天显示出对数期。对混合淋巴细胞培养物进行多参数流式细胞术监测，能够对异种刺激的人类淋巴细胞混合培养物中增殖群体的表型进行分析并评估其活力，从而补充了经典的³H - 胸腺嘧啶核苷掺入试验。这项技术的应用将使人们能够在异种混合淋巴细胞培养物中测量一系列免疫参数。从这些试验中获得的信息对于理解异种细胞相互作用的生物学意义以及监测异种移植患者的免疫状态至关重要。
