利用流式监控高质量小鼠脾细胞的制备  

Monitoring survival of splenocytes and lysis percent of red blood cells by flow cytometry

作　　者：李敏惠[1] 刘阳[1] 杨平[2] 郑静[1] 杨淑霞[1] 阳泰[1] 张露[1] 邹强[1] 李华[3] 

机构地区：[1]成都医学院科研中心,610083 [2]成都医学院生化教研室,610083 [3]成都军区总医院肿瘤科

出　　处：《国际检验医学杂志》2012年第2期136-137,140,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(81072455);四川省青年科技基金资助项目(2010A08-443);四川省教育厅科研基金资助项目(10ZC073);成都医学院科研基金资助项目(CYZ09-020)

摘　　要：目的流式技术监控小鼠脾细胞裂解过程,在确保脾淋巴细胞高活力的同时,充分裂解其中的红细胞。方法以流式细胞术监控ACK和Tris-NH4Cl两种红细胞裂解液的裂解效果,通过细胞融合率判断淋巴细胞活力。结果结合FSC和PI荧光强度分析,在确保淋巴细胞相对存活率大于或等于90%的前提下,ACK处理0.5～1min即可去除90%以上的红细胞,Tris-NH4Cl处理10min也可去除82%的红细胞,获得的淋巴细胞的融合效率为85%～90%。结论流式细胞术能够实时监控ACK和Tris-NH4Cl的裂解过程,获得高质量的脾淋巴细胞以适用于杂交瘤细胞的制备。Objective To development a technique of monitoring survival of splenocytes and lysis percent of red blood cells by flow cytometry.Methods Splenocytes were lysed by ACK or Tris-NH4Cl.The percentage of live lymphocytes and red cells in total splenocytes were detected by flow cytometry.Cell fusion assay was performed.Results The live lymphocytes and red cells could be classified by flow cytometry after dying by PI.If the 90 percent live lymphocytes were need to survivied,splenocytes were treated with Tris-NH4Cl for 10 min to remove the 82 percent red cells or with ACK for 0.5 or 1 min to remove the 90 percent red cells.Using these lymphocytes hybrided with SP2/0-Ag14,the 85 to 90 percent efficiencies of fusion could be detected.Conclusion The progression of lysing splenocyte could be monitorred by flow cytometry to ensure the survival lymphoctytes activity and clearance of red blood cells.

关 键 词：流式细胞术 细胞融合 红细胞裂解液 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]