I'm currently having a problem with clumps of cells after cell permeabilization treatment using 70% ethanol. I want to permeabilize suspension cells and treat with FISH probes. After that, the cells are analysed and sorted with cell sorter and finally embedded in hydrogel for confocal microscopy. The problem is, almost every time after permiabilization, some of the cells clumped. I have tried with cold ethanol (-20) but still. Are there any tricks to prevent this based on your experience or other method which is suitable. I'm combining immunocytochemistry with FISH for cell sorting.
I do lots of ethanol permeabilisation for flow citometry. One thing I saw in a protocol that has helped a lot in reducing cell clumps is to re-suspend the cell pellet in the residual PBS from the previous wash, then add cold ethanol drop-wise while mixing/tapping the tube. Then, mix by pipetting up and down a few times and store at -20C.
I do lots of ethanol permeabilisation for flow citometry. One thing I saw in a protocol that has helped a lot in reducing cell clumps is to re-suspend the cell pellet in the residual PBS from the previous wash, then add cold ethanol drop-wise while mixing/tapping the tube. Then, mix by pipetting up and down a few times and store at -20C.
Thanks Gerson,
I tried the drop wise of ethanol but it did not help much. But putting the whole mix into -20 is new to me. I should try it next time. How long do you store the cells in -20? Usually I just left in in 4 degree for 30min. How about if I add the cells dropwise in the cold ethanol? do you think it will make a difference?
We usually fix the cells in PFA (final conc. = 1%) for 15min in ice, pellet the cells, wash with PBS, pellet again, then permeabilise/fix the cells with EtOH, at least overnight at -20C (they can be left at -20 for days if necessary)... I dunno if adding the cells drop-wise into the cold ethanol would make a difference, but I'm curious to find out! Let me know if it works!
Thanks for the tips! I will try this as soon as I get my samples again. I will inform you when I tried the cell-dropwised method. Thanks again.
Hi Gerson,
I had tested the drop-wise method of cell to EtOH/MetOH or more specifically flushing the cells in the middle of the EtOH/MetOH. After permeabilization, analyzed the cells with flow and found that, it was better or more consistent than dropping the alcohol to the cells. Have to try again and again to be sure. However, I found that the cells tend to adhere to the wall side of tube instead of the bottom during centrifugation with alcohol/higher viscosity solution. Is this normal?
Most protocol suggest 10-15 min incubation in -20 to permeabilized cells. I found that my permeabilization efficiency is lower with 10min MetOH (fix-perm) than EtOH 70% (PFA-fix and 70% EtOH for 1h). Will it be OK increase the time or better change to MetOH-Acetone (1:1)
One more thing, I manage to sort almost 200K cells with Aria II but when I check again, I lost almost 80-90% of cells in the same tube (1mL). Any insight?
Thank you.
That is good news. Did you observe much less cell clumps this way? About using MetOH or EtOH, I don't know, as I've never used MetOH to permeabilise cells. This may be coincidental, but last week I used cells fixed for 3 weeks in EtOH and it was a big mess, even though I was able to gate out cell aggregates/debris and analyse it properly. When I leave cells for 1-7 days in EtOH I observe less "noise".
And yeah, when we do the incubations and wash steps in 1.5 mL microcentrifuge tubes, cells tend to adhere to the walls in a vertical pattern sometimes. It is so annoying. One thing that I do to prevent cell loss is I perform incubations and wash steps in a round-bottom 96-well plate (as recommended by Millipore/Guava) using a plate centrifuge with the brake on low. You prevent cell loss because cells stick to the centre of the round-bottom well, plus you're able to use a multi-channel pipette, which is quite handy.
Good luck =)
Hi Gerson, sorry for the late reply.
I did observed less cell clumps and better visualization whether by FACS or microscope.
I just got some idea of why I had low signals for my probes compare to the antibody. It may involve the ratio of cells and the probe itself in addition to permeabilization efficiency. If this is the case, it should be obvious when I increase the probes conc., which is not preferable (probes is expensive). When you left the cells in EtOH for 1-7 days, does less noise mean more aggregates form or the less noise from the machine?
A plate centrifuge, sadly, we cannot afford to get one. My only problem nowadays are the cell return after sorting is too low. Even though it said I had more than 200K, a second flow analysis show it had less than 50K. it is OK if I just need for observation, but impossible if I want to collect for cell culture (live cell without probes)
Thank you for the insight
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