centrifugation speed/cell prep for flow - (Dec/12/2011 )
Hi all,
I am just wondering if there is a particular significance when prepping cells for flowcytometry to use relatively low centrifugation speed to spin cells down (usually at 300g)? The reason i ask is because I find that I am losing cells over the many times I need to wash/rinse cells centrifuge and aspirate supernatant. I fixed my cells in 1% paraformaldehyde overnight at -20C first and then processing for TUNEL assay. I wonder then if the problem because I did not fix it properly or was it because the centrifugation speed was just not high enough? If anyone has any insight, I'd really appreciate your input!
zienpiggie on Tue Dec 13 04:52:37 2011 said:
Hi all,
I am just wondering if there is a particular significance when prepping cells for flowcytometry to use relatively low centrifugation speed to spin cells down (usually at 300g)? The reason i ask is because I find that I am losing cells over the many times I need to wash/rinse cells centrifuge and aspirate supernatant. I fixed my cells in 1% paraformaldehyde overnight at -20C first and then processing for TUNEL assay. I wonder then if the problem because I did not fix it properly or was it because the centrifugation speed was just not high enough? If anyone has any insight, I'd really appreciate your input!
You might have fixed the cells for too long. I fix my cells with 4% PFA for 10 min, not more than 30 min. Haven't heard of fixation at -20C for PFA because it will solidify in the freezer.
As for washing, 200-300g is sufficient depending on your cell type. Overdoing it will lyse the cells.
I found that washing them in glass flow tubes retain most cells. You can try to wash cells in microfuge tubes so you can monitor any possible cell loss.
Hi science noob,
you are right, I made a mistake recalling what I did. It was fixed in 1% paraformaldehyde on ice for 1 hour, and then after washing with PBS twice was then placed in 70% ethanol and then stored at -20C overnight.
Your flow tubes are glass? Mine looks like it's made of polystyrene. My manual suggests to do my cell prep in the 12 x 75 mm test tubes as the 'polystyrene' tubes would have built up static so cells my adhere to the walls or something. But it does not specifically suggest 'glass'. In that case may be I should prep my samples in the polypropylene microfuge tubes instead then and only move it to the flow tubes on the last stage. Thanks for the suggestion.
Hi,
I am just curious. Why do you store it in ethanol ? why -20C ? I use PBS for storage and place it in 4C. seems fine.
I also store in PBS (with about 2-5% FBS added), at 4C overnight with no problem.
For the centrifugation speed, you don't want to risk damaging your cells, so I wouldn't recommend increasing your speed.
If you are working in a 96 well tray format, you could try gently flicking off the solution between washes? I don't do it that way, but a lab mate does and she swears she loses less cells that way.