Viability and cell cycle analysis of equine fibroblasts cultured in vitro.
This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD((R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.
Lima-Neto
J F
JF
Department of Animal Reproduction and Vet. Radiology, FMVZ, UNESP, Botucatu, SP, 18618.000, Brazil.
Fernandes
C B
CB
Alvarenga
M A
MA
Golim
M A
MA
Landim-Alvarenga
F C
FC
D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D002470:Cell Survival; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000166:cytology*; D005615:Freezing; D016193:G1 Phase; D006736:Horses; D008297:Male; D016192:Resting Phase, Cell Cycle; D016196:S Phase; D013997:Time Factors