I have preparing my PC-3 cells for a cell survival assay. I starve the cells for 16-18 h to synchronize my cell in G0/G1. when I check my cells they are more confluent than before. shouldn't cells stop going through the cell cycle during synchronization? Should i except growth to effectively stop after the 18th hour and not before?
I am also not sure of the starvation period, I have seen longer periods in the literature. can someone please confirm for PC-3 starvation time?
Thank you
Hello,
Serum starvation is usually carried out when the cells are 70 - 80% confluent. You can extend the starvation period to 24 hours. You can even go upto 48 hours. It depends on the cell line.
Best.
Remember that reaching confluency is also a result of spreading of the cell on the substrate, not necesarily mitosis... you can easly check for round shaped cells (most usual mitotic shape) and when you stop seeing those, you are sure thats your starvation time... Also, Malcom has a point, that for most cells when they reach high confluency, contact dependent inhibition could work (depending on the cell line) synergistically with serum starvation for the G0/G1 sincronization.
Agree with both.
I will add that some downstream signalling pathways may remain active. Also, as a compensatory mechanism, some signalling pathways may be activated as a prosurvival response. Also, not all cells will be at the same stage of the cell cycle so you have some heterogeneity. But it depends on the cell line.
Dear Joud Babik
If the goal is to enrich cells in G0 then serum starvation for PC-3 cells will require longer periods and the problem with longer duration is the ability of these cells to form binucleated or tetraploid cells upon stress. I applied 48 hours for observing non-proliferative PC-3 cells. If you want G1/S phase cells, then you can go for thymidine blocks, L-mimosine arrest, Aphidicolin arrest or hydroxyurea treatment.
Serum starvation is the easiest option but you have to be precise in chossing the right confluency of the cells when you apply serum-free media.
You can perform optimization by just a timeline analysis on flow cytometry to see if you observe any enrichment differences in the G0/G1 peaks among different time points.
best of luck
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