Pepsin enzymatic disaggregation of lung carcinoma tissues for flow cytometric measurements

Pepsin enzymatic disaggregation of lung carcinoma tissues for flow cytometric DNA measurement was studied. Cell suspensions were obtained from human primary lung carcinomas obtained at surgery or autopsy. Mechanical and enzymatic disaggregation procedures were used. For mechanical disaggregation, tumor tissue was sliced to 5mm in thickness and minced. The cells were dispersed by forced filtration through a metal mesh. For enzymatic disaggregation, cell suspensions taken after the mechanical procedure were incubated in various concentrations of pepsin for various periods at room temperature or 37°C. Optimum conditions for the pepsin disaggregation method were studied by measurement of changes in CV and peak channel number for G0/G1 peaks by flow cytometric analysis. In this study, optimum conditions for the disaggregation of squamous cell carcinoma and adenocarcinoma were incubation in solutions containing 0.1 to 0.5% pepsin for 10 to 30 minutes at 37°C, and for small cell carcinoma, 0.05 to 0.25% pepsin for 10 to 15 minutes at 37°C.