Use Of The CFDA-SE For Cell Labeling And Establishment Of Flow Cytometry Assay Of Determination Of Cytotoxicity

Objective: To study the methodology for alive cell labeling withfluorescence dye carboxyfluorescein diaccetesuccinimidyl ester (CFDA-SE) andto establish a reliable method to measure cell-mediated cytotoxicity byflow-cytometric assay.Methods:In the study on labeling various tumor cell lines with CFDA-SE,we performed four processes as follows: Firstly, the cells were incubated withCFDA-SE for 8min and the values of fluorescence intensity (FI) weredetermined immediately and during 24hrs after labeling. Secondly, optimalconcentration for cell labeling using the dye was determined. Thirdly, we testedthe toxicity of CFDA-SE fluorescence dye on labeled cells. Lastly, tumor cellswere cultured in presence of the dye for 5 to 15min and then the cells stood alonefor 4hrs until FI was detected, and optimal staining time was analysed byCFDA-SE intensity and cell death rate. In the establishment of flow cytometryassay of determination of cytotoxicity, the test principle was based on target celllabeling with CFSE and subsequent DNA-labelling with PI for identification oftarget cells with compromised cell membranes. Effectors and targets wereincubated at E/T ratios from 100:1 to 6.25:1 at different time points from 1h to6h. We collected 1000 target events and analyzed specific cytotoxicity. Weassayed the expression of CD3+, CD16+CD56+ for both CIK and PBMC.Result: In the study of CFDA-SE for cell labeling, the results show that theoptimal concentration of CFDA-SE fluorescence dye for cell labeling is variablein different cell type, CFDA-SE fluorescence dye is non-toxic to cells since theproportion of cell death caused by CFDA-SE labeling is below 5%, and 8 minincubation with CFDA-SE fluorescence dye is determined as the optimal cell...