Tracking cell cycling activities in RPE-fucci cells

Tracing cell cycling activities in RPE-fucci cells exposed to various FBS concentrations.
Conclusion:
A clear serum concentration-dependent reduction in the proportion of cells arrested in G1 was observed during the cumulative tracing period.
Notes:
Cultures of human retinal pigment epithelial cells (RPE-1) with different frequencies of cycling and non-cycling cells were obtained by applying fetal bovine serum (FBS) at various concentrations, i.e., 0, 0.1, 0.5, 1, 5 and 10%. To quantify the frequencies of cells in different cell cycle stages, RPE-1 cells contained the fluorescent ubiquitination-based cell cycle indicator (Fucci) system. In this system, red and green fluorescence reporters fused to the degradation motifs of the cell cycle-controlled Cdt1 and Geminin proteins mark, respectively, cells in G1 and S/G2, respectively. Simultaneous presence of both reporters mark instead cells in early S phase. After seeding and exposing the reporter cells to the various FBS concentrations, cell cycle analysis was performed at the indicated timepoints (12h, 24h and 48h) by flow cytometry. Cells cultured in normal condition (10% FBS) served as controls for gating cell populations in G2/M, early S and G1, respectively. At least 10,000 viable singel cells were acquired per sample.