一种利用吖啶橙染色快速鉴定重力密度梯度沉降法中生精细胞类型的方法

Acridine orange staining for fast identification of the spermatogenic cells during STA-PUT velocity sedimentation

目的建立一种重力梯度沉降法分离粗线期精母细胞,圆形及长形精子细胞的快速精确的镜检方法。方法利用重力密度梯度沉降法分离,将收集到的每管细胞分别放入96孔板中,每孔加入一定浓度的吖啶橙染液,于荧光显微镜下观察。结果通过分析对应荧光通道下3种生精细胞的细胞核/细胞质染色结果,可快速精确的分辨出粗线期精母细胞,圆形及长形精子细胞。结论建立了一种在重力密度梯度沉降法分离生精细胞过程中快速镜检检测粗线期精母细胞,圆形及长形精子细胞的方法。

Objective To establish a fast and accurate technique of identifying the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation.Methods Using STA-PUT velocity sedimentation method to isolate the pachytene spermatocytes,round spermatids and elongating and condensing spermatids from mouse testes.To determine the cell populations＇ distribution,each tube of cell fraction was then partially transfered to the 96 plate well,and each well was added with acridine orange dye.Then each well was analyzed using fluorescence microscopy.Results Three types of spermatogenic cells can be identified quickly and accurately by it＇s specific cytoplasm/nucleus character using the acridine orange dye staining under fluorescence detection.Conclusions A rubust method to quickly and accurately determine the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation is successfully developed.