摘要
采用0.01MPBS等渗液配制的浓度为70%的乙醇固定液对虹鳟活体胚胎进行固定,并以蒸馏水配制的浓度为70%乙醇固定液作为对照,探讨用于流式细胞术分析的胚胎组织单细胞悬液制备方法。实验结果表明:PBS-乙醇和蒸馏水-乙醇固定液均可获得分散良好的单细胞悬液,但蒸馏水-乙醇固定液制备的胚胎细胞悬液中细胞数量相对较少,细胞碎片明显增多,流式细胞仪检测G0/G1峰较宽。而PBS-乙醇固定液制备的细胞悬液,细胞形态完整、背景较干净,细胞粘连少,几乎没有细胞碎片,流式上机检测各参数值较理想;以PBS-乙醇固定液制备2n虹鳟胚胎单细胞悬液作为材料检测,其结果与以红细胞为对照的样品检测DNA相对含量结果相一致,均为50AU。本研究表明利用PBS-乙醇固定液制备鱼类胚胎组织单细胞悬液方法是可行的。
To probe the better way for the cell suspension preparation which were used in flow cytometry（FCM）,the embryo cells of rainbow trout（Onchorynchus mykiss） were fixed by two kinds of ethanol fixative,the one of which was diluted with 0.01M PBS（PBS-ethanol） and the other was diluted with distilled water（DW-ethanol）,the last concentration of the both were 70%.The results showed that the dispersed cell suspension could be prepared by both of the two methods.The samples fixed by DW-ethanol had fewer cells but a large number of cell debris and wider G0/G1 peak in FCM detection while the one fixed by PBS-ethanol had better cell morphology,cleaner background,less adhesive cells,less cell debris and ideal parameters during FCM detection.Relative DNA contents were detected by FCM,and the results of PBS-ethanol group were the same as the control samples of erythrocytes from diploid rainbow trout.Although the CVs of the former were wider,they were all in an acceptable range.It is feasible to get cell suspension of embryos as flow cytometry sample by fixative of PBS-ethanol.
出处
《水产学杂志》
CAS
2010年第3期40-43,共4页
Chinese Journal of Fisheries
