Cell synchronization of Drosophila cell line
Cell synchronization of Drosophila Kc cells
Kc cells were cultured in 15-cm plates at a density of 1×106cells/mL in Schneider’s Media (Invitrogen 11720034) supplemented with 10% FCS and penicillin/streptomycin. The cells were arrested in G2 by the addition of the hormone Ecdysone (Sigma H5142, 蜕皮激素, C27H44O6) at a final concentration of 1.7 µM. To arrest the cells at the G1/S transition, the cells were released from Ecdysone into 1 mM HU. For replication timing, cells were pulsed with 50 µg/mL BrdU (Roche 280 879) for 1 h at either 0 or 6 h following release from HU. For identification of early origins, cells were released from G2 into medium containing both BrdU and HU at the same concentrations as above. Cell cycle progression was monitored by FACS.
Molecular formula of Ecdysone
Cell synchronization of Drosophila S2 cells:
Briefly, log-phase (2x106/ml) cells were first incubated with 0.2 nM ponasterone (甾酮A, C27H44O6) A for 24 h to obtain G2 cells. Cells were then rinsed with phosphate-buffered saline (PBS) three times, resuspended in fresh Schneider’s Drosophila medium (GIBCO) containing 5% each of Fetal Bovine Serum and Bovine Calf Serum (JRH Biosciences), along with 1.5 mM hydroxyurea (HU, 羟基脲, CH4NO), and cultured for 18 h to obtain G1/S cells. Afterwards, these cells were rinsed with PBS three times, cultured in the above-specified medium without HU and harvested at various time points for analyses.
Molecular formula of ponasterone
BrdU-FACS or FACS analyses :
Cells grown in 6-well plates were treated with BrdU (10 μM) for 45-60 min, fixed with 80% cold ethanol, treated with 3N HCl, washed and incubated with anti-BrdU MAb (Becton Dickinson) for 60 min. After washing, cells were incubated with Alexa Fluor ® 488 goat anti-mouse IgG (Invitrogen) for 30 min. These cells, or ethanol-fixed cells (for direct FACS assays), were treated with propidium iodide and RNase A for 30 min before FACS analyses.
Reference:
Lee et al.,2010, JBC. Drosophila Octamer Elements and Pdm-1 Dictate the Coordinated Transcription of Core Histone Genes.
David M. MacAlpine., 2004, Genes Dev., Coordination of replication and transcription along a Drosophila chromosome.。