We have been trying to reduce levels of pVEGFR3/pERK by serum starving our LECs (LONZA) for 16 hours. Although this time point had been very succesful in reducing levels of pVEGFR2/pERK in HUVECs, we cannot seem to reproduce this in LECs. Does anyone have any tips/advice for doing this in this very difficult cell line?
Hi,
I would suggest to do starving time point, starting from 16, 24, 48 hours and see what the best time to reduce the signal, for me I used 24 hours to starve my cell but that was for erbb2 an erbb3.
Hi,
I assume that you use a serum-*reduced* medium? Our experience based on several primary epithelial and fibroblastoid cells showed that residual FCS serum concentrations to achieve reliable G0-arrest (only cells with G1-DNA content peak in the flow cytometer) ranged from 0.5%-0.2% and the time from 24-48 hours.
In addition, confluency had a large impact. Some of the more stubborn fibroblast clones (MEFs in early passage) would continue to proliferate for days - albeit slowly - in 0.3% serum when plated at ~ 25% confluency. A mean starting condition was about 0,3% serum, 36 hours, 50% density.
Best wishes, Ernst
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