I work with suspension cells, specifically, THP-1. I counted the cells before centrifugation ( directly from the flask) and after centrifugation (pellet). Before I had 10 million cells and after I had 4.8 million cells.
a) Why did this happened? What happened to the rest of the cell
Centrifugation was at 1,580 rpm x 5 minutes.
b) Does the size of the falcon tube matters? I used a 50mL falcon tube to hold 20mL of culture media.
c) Instead of discarding the supernatant, I transferred the supernatant into another tube (just in case) and centrifuged that tube and there was no pellet or cells present.
a) did you check the flask they came from? Even suspension cells can stick loosely to the surface of flasks: I usually wash 1x with media to collect any reluctant cells.
Also, centrifugation speed is meaningless without knowing the radius of your rotor: can you give the value in rcf (i.e. g)?
b) no
c) probably no cells, then.
Honestly, the fact that your second measurement is almost exactly half your first makes me wonder if you've simply made a calculation error (for instance, resuspended your cells in double the media volume before counting, but calculated total cells based on the original volume).
Unless the cells have died and burst to debris during the process, you should have intact cells in the suspension or in the pellet:
a) When you count suspension cells, did you disperse the cells well and counted the number multiple times? Sometimes, you get more cells when you use just one time small volume for counting the suspension cells.
b) I'm not sure which suspension media you use. I assume it's a liquid type, not a sol-type. Bigger volume requires longer centrifugation time with the same g force.
c) Did you look at the supernatant after centrifugation under microscope? It should have nothing or only cell debris.
Its normal to count less cells, but not half.
You should use 300 g for centrifugation and maybe 10 minutes for a large volume, 5 minute might not be enough.
However I agree with john that the difference is probably exaggerated by a calculation error, especially if there were no cells in the supernatant.
Thank you all for your answers. The 1,580 RPM is equivalent to 530 g after using a rotor calculator and measuring the rotor radius. 530g x 5 minutes is too much? Would that have destroy the cells and the ones that survives are found in the pellet?
Indeed, cells were disperse to promote equal distribution along the media.
Didn't observed the supernatant through the microscope, however, I counted multiple times with the supernatant and it was rare to find a cell.
500g should be fine. 500g for 10mins is better than 5mins, but 5 should be adequate.
How are you calculating your cell totals?
1) Before centrifugation, I would count the cells by resuspending the total media in the flask with a disposable pipette and transfer a small aliquot to an eppendorf tube. From that eppendorf tube, I take out 10uL of cells and resuspend them with 10uL of Trypan blue to make a dilution factor of 2 and count with the hemacytometer.
Results were: 121 viable cells/ 1 dead cell across all 4 quadrants.
I calculated total ammount of cells in my flask by:
121 viables cells/ 4 quadrants x DF (2) x 10^4 x Volume in flask (20mL) = 12,100,000 cells in total.
2) Subculturing was done by adding 17.2mL of fresh RPMI 1640 media to a new T-25cm flask and original flask had its media resuspended before taking out 2.8mL of media (to seed 1.7 million cells) to the flask to make the final volume of 20mL. This should leave me, in theory and approximately, with 10.4 million cells for freezing.
3) After centrifugation, I would count the cells by resuspending the pellet with 1mL of RPMI 1640 media and, in the same matter as above, i tried counting with a dilution factor of 2 but there were too many to count and I would take forever. I decided to raise the dilution factor to 10 (90uL of trypan blue and 10 uL of cells).
Results were: 194 viable cells/ 4 dead cells
I calculated total ammount of cells in the 1mL of media by:
194 viable cells/ 4 quadrants x DF(10) x 10^4 = 4,850,000 cells in total.
Maybe I should have left it for more than 5 minutes?
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