I am culturing lymphocytes and they tend form visible clumps.. Does anybody know how to avoid this and if this is common ? I am new in culturing them. Thanks
This is entirely normal and your cells are growing as they should do . Do not worry. When the medium turns yellowish it is time to split or feed them again
Thank you. So, when I split them, I centrifuge and resuspend the cells. I try to break the clumps by pipetting. Is this okay ? Not all clumps break though. My follow up experiment is Immunofluorescence, so I am wondering how to make them into a single cell suspension before putting them on the coverslips.
If you are splitting just so that you can grow more cells then just pipette up the cells and some old growth medium and transfer it to new flasks without centrifugal ion and shake gently to break up the clumps. I am not sure about the best way to generate a mono layer as I only grew bev line to make DNA so clumping was not an issue but shaking for a few seconds will break up most of the clumps of cells. Hopefully someone can advise on a better way of getting single intact cells
Thank you. Shaking did not help. I had to centrifuge and break the clumps using a pipette and this helped but as it started to divide, it started clumping again.
clump formation is normal and is how ebv transformed cells grow. If you need a monolayer I think you will have to break up the clumps , drip the cells onto a slide and use another slide end to smear the cells onto the first slide by dragging the spreader slide over the target slide to spread a layer of cells onto the target slide then fix them onto the slide in a suitable manner...heat, alcohol or whatever is appropriate to your technique. I do not think that you can grow them on a slide or cover slip in the same way as fibroblast lines
I found a way to fix them on the slides. Centrifuge and resuspend in PFA, wash, and centrifuge them onto cover slips. This works.
excellent. I hope the immunofluorescence expts go well Shivasankari. Good luck.
Paul
- Badges
- Science method