Hello everybody, i have a question about cell fixation for flow cytometry analysis (i am trying to optimize fixation for pH2ax analysis).
I fixed my cells with 70% Ethanol following this protocol:
Cell Fixation Using 70% Ethanol
- Prepare 70% Ethanol (dilute with H2Ob.d.) and chill to -20°C.
- Prepare target cells of interest and wash 1X with PBS, centrifuge at 1000rpm 5’ minutes.
- Discard supernatant and loosen the cell pellet by vortexing.
- Add 3ml cold 70% ethanol drop by drop to the cell pellet while vortexing.
- Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour.
- centrifuge 1800rpm 15’.
- resuspend the cell pellet in cold TBS, wash once and acquire.
The problem is that after this fixing the cells appear really different from the not fixed ones and i am wondering if this is normal or not..i read that cells could change their FSC/SSC after fixation but i don't know if this is the case. I attached the pdf with the plot of viable and fixed cells.
Thanks to all in advance!!!
Dear Marta Canta , Your concern is very valid. After Perm/Fix FSC/SSC scatter do change for which you need to adjust voltages accordingly. But after only fixation, i did not find much difference in scatter.
I am not sure about the protocol you are using, but we preferentially do not vortex cells after/during adding fixative. We first resuspend the pellet in residual volume by vortexing and then add the fixative (We use PFA) and mix by inverting the tube gently.
All the best.
Dear Jitendra Kumar Shandilya, thanks a lot for your answer! i will try following your protocol.. maybe i could also resuspend pellet in PBS and then add ethanol to reach 70%..
unfortunately i cannot use PFA at the moment!!
Thanks a lot
All the best.
Il a peut être une relation avec le solvant polaire ethanol peut on utilise autre exemple toluène
Hi Marta Canta
1. Resuspend the cells in 1 mL PBS and transfer them to 15 mL falcon tube.
2. Add 4 mL of 96% ethanol drop by drop on vortex (it will make final ethanol concentration ~75%)
3. Leave the cells on -20 degree overnight (at least for 6 hours)
4. Centrifuge and remove the ethanol
5. Wash with PBS once
6. Proceed downstream (for example RNAase treatment and PI staining for cell cycle).
"Dissociated cells were fixed and permeabilized by adding an equal volume of ice-cold ethanol to the above cell suspension for a final concentration of 50% ethanol and kept on ice for 15 min with occasional shaking."
https://pubmed.ncbi.nlm.nih.gov/23895375/
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