Hi all,
I'm currently facing issues in the number of cells retained following ethanol fixation. I'm seeding 700,000 22RV1 cells per T25 flask.
Before fixing, there is a cell pellet present at the bottom of the tube. I know that ethanol fixation causes the cells to become almost translucent and after fixation + PBS washes, I can see that are cells present in the flow tube.
However after fixation, even just left overnight, when I spin the cells down there seems to be very little left. When I stain them and read on the flow cytometer, I'm not even getting 10,000 events before the whole sample is gone, and even less cells!
Edit: I found that the storage step was where I was losing cells! I was storing the fixed cells in 70% ethanol at 4°C. I changed to storing the cells at -20°C and I had a high quantity.
Hello Amy Kinsella ,
Maybe you could try to do the fixation again (T) along with a control lot (C, treated with PBS, not ethanol) and then resuspend the cells, take half of the volume (V1/C1) and analyze it by flow cytometry immediately. Leave the other half (V2/C2) overnight just as before and analyze them identically the next morning. If V1 and V2 have similar behaviors, then your problem is not time dependent, so you can focus on what's technically wrong with your fixation reagent or even with your cells. If V1 acts normal and V2 is bad, then it's a time dependent effect and you should focus on what's happening overnight. If C1/C2 acts normally, while cells are gone with V1 and/or V2, then you should investigate whether your fixating agent is ok.
I'm really curious about this. Hope it helps!
Ion Bogdan Mănescu Many thanks Ion.
I've previously read these cells for unfixed and re-suspended in PBS and there were no problems with reading 10,000 cells/35,000 events! After fixing, the cells are stored in PBS at 4°C.
I'll try that if my experiment doesn't work again this week!
Hi @Urs Mörbe, I've been using ethanol to fix due to prior in-house optimization using the protocol I'm following now.
I'm planning to change some of the steps in the protocol this week to see if it will solve my problem.
If it doesn't work out, we're considering using PFA or methanol instead!
Amy Kinsella how long is your centrifugation? I had similar problem with methanol fixation - after standard 5 mins run I lost most of my cells. Centrifugation for 10 mins solved the problem.
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