I am having some difficulties with my 22Rv1 cells. I am finding that a lot of debris accumulates in my plates. Washing with DPBS only partially helps. Also, dissociation from the plate can take up to 10 minutes (I use 1ml Trypsin-EDTA 0.25%). And, my greatest difficulty is that the cells seem to aggregate after trypsinizing, which makes cell counting (for cell viability assays) a real challenge. Has anyone ever encountered these issues with their 22Rv1 cells? Would anyone have any recommendations for me? I grow my cells in RPMI 1640 media with 10% HI-FBS, L-glutamine 2mM, and Pen-Strep 1X. I replenish media every two days and split every 5 days. Thank you kindly.
Hello
Cell clumps form when
1.cells are over exposed to trypsin during trypsinization.
2. cells are overgrown at the time of subculturing because DNA released from dead cells in culture due to its sticky nature causes clumping of cells and debris.
I would suggest that you rinse the cells in Trypsin-EDTA for a few seconds and then expose the cells to trypsin-EDTA for not more than 5 minutes. Tap the plate from the sides if cells are still loosely attached.
Subculture the cells when they are 80% confluent. Do not wait for 100% confluency. This might help reduce the cell clumps.
By pipetting the cell suspension up and down few times you can get rid of small clumps.
I hope this is helpful.
Good Luck.
Thank you, Malcom and Arje. I cut down the contact time with trypsin and split at a lower confluency (~70-75%). This helped enormously. Thank you, Malcom for the advice.
Maybe cutting down the trypsinage duration works, also transfering the cells to a larger T-flasks works if you are doing suspension cell culture, also I am thinking of lower counts of initial seed in the initial RPMI media, Optimizae the glutamine concentration as well, hope this works, best,
Maybe cutting down the trypsinage duration works, also transfering the cells to a larger T-flasks works if you are doing suspension cell culture, also I am thinking of lower counts of initial seed in the initial RPMI media, Optimizae the glutamine concentration as well, hope this works, best,
Maybe cutting down the trypsinage duration works, also transfering the cells to a larger T-flasks works if you are doing suspension cell culture, also I am thinking of lower counts of initial seed in the initial RPMI media, Optimizae the glutamine concentration as well, hope this works, best,
Maybe cutting down the trypsinage duration works, also transfering the cells to a larger T-flasks works if you are doing suspension cell culture, also I am thinking of lower counts of initial seed in the initial RPMI media, Optimizae the glutamine concentration as well, hope this works, best,
Maybe cutting down the trypsinage duration works, also transfering the cells to a larger T-flasks works if you are doing suspension cell culture, also I am thinking of lower counts of initial seed in the initial RPMI media, Optimizae the glutamine concentration as well, hope this works, best,
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