A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle

Abstract

Objective: To set up a three-parameter method for cell cycle analysis by two-laser flow cytometer, which can detect two types of cyclin plus DNA content in one measurement, and that analyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysis of two types of cyclins and DNA content simultaneously in individual cells by two-laser flow cytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. A Triton-X100 permeabilization procedure was optimized for detection of two types of cyclins. One cyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other, indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochrome DAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method. Results: Permeabilization with 0.5% Triton-X100 in PBS containing 1% BSA for 5 min on ice provided optimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It was found that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured with no compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growing MOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously, allowing for analysis of cell cycle phase specific perturbations without the necessity of cell synchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine and cyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleled expected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only be applied to measure unscheduled vs. expected cyclin expression but may also be used to estimate the fraction of cycling cells in up to 6 cell populations.