Hi, I'd like to have some suggestions on isolating proliferating cells from a mixture of senescent cells and proliferating cells culture.
I am planing to isolate the proliferating cells, because the cytokines secreted by senescent cells will affect their growth. SSC/FSC would be an applicable way, since senescent cells are much bigger, but I am wondering whether there is some more efficient option such like staining.
@Bin Yue,
senescent cells are, indeed, much bigger than their proliferating counterparts so you can try to sort them out using forward scatter. My advice to you is, however, to check after the separation the presence of SA-b-Gal-positive cells in "young cell" population, as often senescent cells expressing SA-b-Gal are of normal size. You can also used fluorescence for gamma-H2A.X foci - non-hypertrophic senescent cells will express high level of fluorescence.
Good luck
I have no answer for you but you might contact/locate Dr. Jules Selden who has considerable experience with cell sorting techniques.
Hi Bin,
You should find a proliferating/senescent cell marker (positive or negative selection) and, as Paul said, consider cell sorting (FACS). Another possibility is using magnetic beads conjugated with an antibody against your selected marker (easier to maintain sterility)
Good luck!
Thank you, Krzysztof and Martin, The FSC method is the only one I can imagin. The sabetaGal or gammaH2AX or any staining afterwards aiming to discreminate the senescent and proliferating cells is not the point. The point is what marker you can use for isolation in mild conditions. Unfortunately there is no specific marker on senescent cells except sabetagal, which requires harsh treatment for cell.
I am thinking about using Edu or Brdu involvement, but I don"t know whether this could be done while keeping cells alive.
I understand. Another alternative, much labourious, it's to transfect cells with the marker gene with GFP (for example). In this way you can combine the size criteria and marker expression in your gating strategy, obtaining higher purification yields of the population of interest
Best of lucks!
Bin Yue,
as per senescent cells, their isolation according to hypertrophy (forward scatter) is the only way available. SA-b-Gal and/or g-H2A.X that I've suggested previously follow cell sorting in order to confirm that you really got a pure population of young cells.
In the absence of a specific cell surface marker which could discriminate proliferative versus senescent cells, indeed, poses a challenge. Overall size of cells under these two growth conditions have not been reported to be significantly different. However, use of antibody to Brdu or Topoisomerase II alpha are well known markers of proliferative cells. These antibodies could be used to assess the purity of post isolated cells.
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