Good day, I have been having problems with cell counting with THP-1 cells. I counted my cells directly from the flask by resuspending the culture media to promote equal distribution of cells throughout the flask and proceeded to count the by taking a small aliquot of the media. I counted 121 viable cells and 1 dead cell for a 99.1% viability and a total of 12 million cells. I then seeded the volume containing more or less 1.7 million cells in a T-25cm flask with fresh media.
My cells remaining should be 10.4 million cells, which I wanted to freeze them. I centrifuged the remaining culture media at 1,580 RPM x 5 minutes and it resulted into a pellet. However, I wanted to count the cells present in that pellet and I resuspended the cells in 1 mL of culture media and my total concentration was 4.8 million cells. They were very viable (more or less 98% but with less cells).
Why were there so much less cells after centrifugation? There were 4.8million cells/ 10.4 million. I could only freeze 1 cryovial instead of 2.
I didn't aspirated the supernatant, I centrifuged the supernatant just in case the cells were still there and no pellet was seen.
Hello,
You can try to count the seeded cells from T-25 flask. Maybe this will help you to understand and to find out what really happened with the cells.
Thete is always cell lost by centrifugation. The more the cell number usualy the less is the loss.
Yours are around ten millions. Usualy not many will be lost.
Cell lost can be due to handling when removing the supernatant. When you centrifuged the supernatant you did not see the pellet, but if you resuspend you may get some cells.
Other possibility is your centrifuge does not work well and many cells were not pelleted yet.
Another possibility, you took more cells when you seeded. This can happen if you did not resuspend well, or you work slow, so the cells settled down and you took the bottom part where the cells were more concentrated. If this happened you can checked the flask. It will become confluent faster as you seeded more.
Hope you get good result next time.
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