Cell cycle analysis of bovine cultured somatic cells by flow cytometry.
This study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells (fetal fibroblasts, adult skin and muscle cells, and cumulus cells) after culture under a variety of conditions; 1) growth to 60-70% confluency (cycling), 2) serum starvation, 3) culture to confluency. Cell -cycle phases were determined by flow cytometry with propidium iodide staining enabling the calculation of percentages of cells in G0 /G1, S and G2 /M. The majority was in G0/G1 regardless of cell type and treatment. Serum-starved or confluent cultures contained higher percentages of cells in G0/G1 (89.5-95.4%; P < 0.05). Percentages of cells in G0/G1 increased as cell size decreased regardless of the cell type and treatment. In the serum-starved and confluent cultures, about 98% of small cells were in G0/G1 . Serum-starved cultures contained higher percentages of small cells (38.5-66.9%) than cycling and confluent cultures regardless of cell type (P < 0.05) . After trypsinization of fetal fibroblasts and adult skin cells that were serum-starved and cultured to confluency, the percentages of cells in G0/G1 increased (P < 0.05) on incubation for 1.5 (95.7-99.5%) or 3 hr (95.9-98.6%). These results verify that serum starvation and culture to confluency are efficient means of synchronizing bovine somatic cells in G0/G1, and indicate that a more efficient synchronization of the cells in G0/G1 can be established by incubation for a limited time period after trypsinization of serum-starved or confluent cells.
Cheong
Hee-Tae
HT
Department of Veterinary Medicine, College of Animal Resource Science, Kangwon National University, Chuncheon 200-701, Korea.
Park
Tae-Mook
TM
Ikeda
Koji
K
Takahashi
Yoshiyuki
Y
D000818:Animals; D002417:Cattle; D002453:Cell Cycle; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005260:Female; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry / Q000662:veterinary*; D008297:Male; D032342:Muscle Cells / Q000166:cytology; D006080:Ovarian Follicle / Q000166:cytology; D012867:Skin / Q000166:cytology
D016895:Culture Media, Serum-Free