Hi, I just have general question about passaging cells.
I have two t75 flask of Tramp C1 culture, and when I am subculturing them, after all the necessary steps, I get 5 mL of media+trypsin from each flask into a 15mL conical tube. Then, I centrifuge that 10 mL cell solution, and re-suspend the cell pellet with fresh 5mL media.
Then, I count the cell from 5mL cell solution with automated cell counter, and get live concentration of 5.2e6 cells/mL.
If I am trying to calculate the total live cell concentration, do I multiply 5.2e6 by 10mL(volume before centrifuging) or 5mL(media volume used to re-suspend the cell pellet after centrifuging)?
Also, if I want to aim to have 5e6 cells/mL per t75 flask, how much cell volume from 5mL cell suspension should be put into new t75 flasks with 10mL fresh media? Also, how many new t75 flasks should be prepared for this passage?
I am fairly new to this cell passage and very confused in figuring out how many new flasks I should prepare if I am subculturing from two old cell culture flasks. Please help me out!
Hi Joseph,
Concerning your cell concentration:
If you count your cells in a final volume of 5 ml, you must multiply your cells/ml by 5 (in this case 5.2e6 cells/ml x 5 ml = 26e6 cells in total) and not by the volume you used before.
For your new passage I would generally say, prepare what you need and freeze the lower passages for later use. So let's say you need 20e6 cells for your experiment, according to your data one T75 flask should be sufficient.
I assume you want to seed a total of 5e6 cells in your new flask and not 5e6 cells/ml? In this case you only have to calculate how many cells you have to take from your 5 ml cell suspension and seed them into your new flask. For example, here you have 5.2e6 cells/ml; that means for 5e6 cells (5/5.2 = 0.961) you have to take 961µl and transfer them into your new culture flask. Now you only have to fill up to 10 or 15 ml with fresh medium.
Hope this was helpful.
Best
Hi, Maria-Bernadette Madel
Thank you for your answer.
If I count the cells from 10 mL solution from old flasks, before centrifuge and re-suspension with 5 mL of new media, then I should work with the cell count calculated from 10 mL when seeding or preparing for experiment? I was thinking so because the cell number wont change after centrifugation and re-suspension with 5mL of new media.
Am I right? I thank for your help.
Hi Joseph,
You're absolutely right.
My mistake, I thought you centrifuged and then counted. If you count your cells from 10 ml you have to multiply of course by 10. The quantity of your total cells remains the same after centrifugation and resuspension in any volume.
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