Hello, I'm working with the THP-1 cell line. This is my first time culturing suspended cells. I lost one vial from ATCC, and they explained that the cells died due to over spinning. However, I was spinning at 200g for 10 minutes. For the next batch, I'm thinking about not spinning and instead separating the cells for subculture. For cell counting, I attempted to allocate 1ml from 10ml after mild resuspension, followed by 10 micro/L. However, the issue is with a tiny number of cells. Even 2-5 cells in one quadrant.
My question is
1. what will be the procedure to allocate the cells for counting?
2. For hemocytometer which equation should I use?
Please help me with valuable advisees.
Hello Chowdhury Forhadul Karim
1. what will be the procedure to allocate the cells for counting?
Upon reaching confluency, THP-1 cells may be split every 2–3 days at a ratio of 1:5 to prevent over confluence or to stop cell density exceeding 80%. Spinning THP-1 cells can cause stress as the cells will be subjected to centrifugal force. You may centrifuge the cells at 1000-1200 rpm for 5 mins once a week, which is acceptable. After centrifugation discard the supernatant and tap the pellet. Add 1 ml of complete media to the cell pellet, and take 10ul of cell suspension to which you will add 10ul of trypan blue. Mix and take the cell count using the hemocytometer.
2. For hemocytometer which equation should I use?
Use the following equation.
Total cells/ml = (Total cells counted x Dilution factor x 10,000 cells/ml)/ Number of squares counted
So, for example, if you dilute your cell suspension 1:1 with trypan blue (dilution factor will be 2), and if the total count is 300 cells in the four corner squares (number of squares counted will be 4), then the total cells/ml will be
Total cells/ml = (300 cells x 2 x 10,000 cells/ml)/ 4 = 150 x 10^4 cells/ml = 1.5 x 10^6 cells/ml
Best.
That customer support rep is just talking spouting out nonsense to make you go away. Overspinning does not sterilize your culture. Certainly 200g is safe.
It was something else you did though. Try to find out what :) Because you kind of need to know for next time. Watch everything attentively. Killing all the cells takes a lot of firepower. Somewhere you're doing it.
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