I am evaluating the uptake of florescent nanoparticles in a cancer cell line. After application of the nanoparticles, washing and harvesting the cells, do you think it is appropriate to fix the cells using 70% alcohol for example before analysis with flow cytometry?
One may ask why? simply because the flow cytometer is located far away the culture unit and it may take few hr to start analyzing the samples. One may think of a flow cytometer with heavy schedule as well.
I do not intend to sort my cells for sure.
Also could this affect the side scattering of the cells?
Ethanol fixation for flow cytometry¶
Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom
Fixation for flow cytometry with ethanol. This method is very simple, quick and allows samples to be stored for weeks at –20°C.
- From a 35mm-10cm plate harvest cells with trypsin into a 15ml tube.
Keep media as this helps to inactivate trypsin and many mitotic and dead cells will be floating in the media
- Pre-chill 100% -20°C high grade Ethanol
- Centrifuge at low speed 1500rpm 10 minutes to pellet
- Remove supernatant media and resuspend in 1ml PBS
- Transfer to a 1.5ml eppendorf
- Centrifuge at 5000rpm for 2-5min to pellet
- IMPORTANT: Remove supernatant PBS and gently resuspend pellet in 300ul of PBS
- Add 700ul of 100% -20°C high grade Ethanol (EtOH) inverting tube a few times to ensure proper mixing and a good fixing of cells.
- Store at –20°C until required for flow cytometry/FACS
It is ok to fix your cells before analysis with flow cytometry. However, I strongly advise to test different fixation methods and opt for the one giving better results. Ethanol is not just a fixative, it will also permeabilize your cells, so you could end up loosing your nanoparticles through the holes before enough cross-linking is achieved.
Fixation will definitely affect side-scatter, as dead cells have lower forward-scatter and higher side-scatter than living cells. However, if your controls (including unstained control) are treated the exact same way as your samples, there is no reason why this should affect your results. Good luck!
We usually suspend the cells in 200uL PBS with 1% BSA, and then add 100 uL of 4% formalin in PBS. We get robust results when measuring the uptake of nanoparticles.
You could fix your cells before flow cytometry. We usually stain the cells, fix them and then do flow cytometry the next day. Samples are usually fixed with formaldehyde, not ethanol. Several suppliers sell flow fixative solutions and they come with directions for use. You could also make your own if you have access to formaldehyde. See the link below for an explanation on why formaldehyde is better than ethanol.
https://bitesizebio.com/22141/fixation-and-flow-cytometry/
Yes, you can fix the cells before analysis in a flow cytometer. We use 1% Paraformaldehyde in PBS for fixing the cells.
Thank you all for replies. This is so helpful.
For anyone having a similar issue, I conclude it is best to use paraformaldehyde rather than alcohol for this purpose.
Again, thank you all.
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