一种基于荧光强度分析的高通量定量检测细胞凋亡方法

A High-throughput and Quantitative Assay Based on Fluorescence Intensity for Detection of Apoptosis

根据正常细胞、凋亡细胞和坏死细胞的细胞膜对核酸荧光染料的不同选择通透性,用4μmol/LYO-PRO-1(YP)和4μg/ml碘化丙啶(Propidiumiodide,PI)染色96孔板中的细胞样品。分别在485/538(Ex/Em,nm)和530/590(Ex/Em,nm)的检测波长下借助荧光分光光度计检测细胞样品孔的YP和PI荧光强度。将YP和PI荧光强度值与用荧光显微镜对同一细胞样品细胞凋亡和坏死的定量分析结果相对应,通过对YP荧光强度值与凋亡细胞数的直线回归分析(r=0.999,P<0.01),得到依据YP荧光强度值求得凋亡细胞数的直线相关方程。该方法可检测出样品中少至180个的凋亡细胞,具有灵敏度高和快速高效的特点。

Based on the different permeability of DNA-intercalant dyes YO-PRO-1 （YP） and propidium iodide （PI） to the membrane of viable, apoptotic and necrotic cells, cell samples were stained with 4μmol/L YP and 4μg/ml PI for 10 min, and the fluorescence intensity of both YP and PI were measured by fluorometer at Ex/Em wavelength of 485/538nm and 530/590nm, respectively. The correlation between YP fluorescence intensity and the apoptotic cell number was confirmed by fluorescence microscope and linear regression （ r=0. 999,P 〈0. 01 ）. As a result, the equation that accounted for the dependence of apoptotic cell number on the fluorescence intensity of YP was derived. This method proved highly sensitive, as it was able to detect as few as 180 apoptotic cells in a sample. Furthermore, apoptosis detection could be easily and quickly carried out in 96-well plates, which made it suitable for high-throughput applications.