Hello, I want to perform FACS analysis to assess the cell cycle status in specific bone marrow cell populations by looking at the Ki67 against DAPI signal. We have working antibodies and DAPI solutions and I am troubleshooting the protocol.
In our last experiment, in one of our samples we observed a granular cell population that is DAPI high (comparable to signal in G2/S/M) but Ki67 negative/low (comparable to signal in G0). I know that all cells between G2/S/M phases according to DAPI signal should also be Ki67 positive/high.
My question then is, has someone experienced something similar while testing the protocol? I am wondering if it is a technical issue (eg buffer composition, DAPI concentration, machine voltages) or if this population can indeed come up in some cases. I wasn't able to find much information by quickly browsing the literature.
Thanks everyone!
I have experience with ki67 antibodies. There are several things I found for myself:
1) ki67 antibodies produced by different companies are different, and some may not be reliable, so it would be nice to confirm that your antibody is actually working as intended.
2) Some cells do not express ki67 at expected cell cycle phases (various progenitor cells give ki67 signals in unexpected phases, for example).
Thanks for the answers!
@Yulia that is a valid point regarding the antibody. We are also still titrating the concentration to find our what works best. I also didn't know that progenitors can give this confusing signal, I will go back to my gating and make sure I am not including unwanted populations.
@Han we are using DAPI to assess DNA quantity and distinguish between G0-G1 and G2-S-M phases and simply gate out dead cells in our analysis. All cells are permeabilised so DAPI will be able to access the nucleus for all of them anyway. But adding another viability dye may help out with the gating, and maybe what we are seeing is indeed noise so I will keep this in mind.
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