My BV2 cells have been thawed for almost two weeks now and are in their third passage since thawing. I have plated them in a 24-well plate at a density of 75k cells per well, and they are well distributed throughout the wells, however, I am having an issue where clumps of round-shaped BV2 cells are gathering at the center of the wells.
Prior to plating, I checked for cell viability and found that over 99% of the cells were living after trypsinization (2ml trypsin EDTA in a T75 flask for 7.5 min followed by a 3:1 addition of ES to trypsin EDTA to neutralize the reaction) on their third day in their second passage since thawing. We were encountering this issue before and tried to ameliorate the issue by triturating the resuspended cells (after 5 min centrifugation at 1000 rpm, resuspended in DMEM [GlutaMAX, 10% FBS, 1% P/S]) through a 30-gauge syringe needle then using a 40 µM cell strainer to strain out clumps of cells without significant success. There are still clumps of BV2s, just as there were without such trituration and straining. We have tested this at different seeding densities and with/without PDL in the plate with no success...
We are now at 48+ hours in vitro without any significantly positive changes to the condition of our cells.
Any ideas? Any similar experiences?
Best,
Maxwell Foote
The eternal struggle of the clumping cells in 24 well plates. I just wrote an answer on another question with a similar problem. I'm not familiar with BV cells but clumping seems to be especially an issue in smaller wells. Possible solutions- plate in a larger plate (10cm plates are perfect for this), if cell numbers allow. I think whats happening is that the cells vibrate and swirl in the media and settle in the middle of the plate. Even if cells are a bit clumped in the seeding solution it shouldn't make them plop down in the middle of the plate. Also annoyingly some cells will clump even more when they are stimulated.
After you seed have a look down the microscope, if they look even then leave them on a table for 10 mins without any vibration from a centrifuge etc. Then carefully put them in the incubator. Avoid banging the incubator door when opening and closing. Every 20 mins push the plate forwards and back a few times and then side to side a few times- this helps them even out. Periodically have a look down the microscope to check they look even after this. I hope it helps- its worked for me recently.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line