Hi all,
I am trying to wrap my head around splitting cells into different surface-area flasks. I could really use your help! I currently have 8 T-25 flasks of HEK-293T cells. I am familiar with splitting from a T-25 to T-25 or a T-75 to T-75. However, at some point, I would like to split perhaps 4 of the T-25 cells into T-75 flasks. Is that possible? If yes, how would the dilution be? A detailed explanation would be really helpful! Thank you in advance:)
Best Regards,
Mathangi
Hi Mathangi,
Yes it is absolutely possible to split cells from a T-25 to a T75 and even to a T175, if needed. To answer the question of how much cell suspension to take to passage from a T25 to a T75, depends on the type of cells, their growth rate and in how much time you want the flask to be confluent for the next passaging.
In your cause I would recommend you to just take 3 times the amount of cells you usually take to split cells from a T25 to a T25, as a T75 is 3 times larger than a T25. Hope I could help.
Hello Mathangi
What Sidrah has mentioned is absolutely right. Since T-75 is three time larger than T-25 you should seed three times that number. I usually seed 0.2 million in a T-25 flasks. So i will seed 0.6 million cells in my T-75 flask. Other factors also matter like the cell type and doubling time. All the best.
Thank you so much Sidrah Chaudary and Malcolm Nobre ! This information is useful!
May I clarify if this would be the right procedure?
1. After washing, trypsinizing and neutralising the cells in the T25, I re-suspend them in 10mL of media.
2. Thereafter, I take 2mL of the suspension and add to 10mL media in the T75 flask. (for a 1:5 dilution)
Supposing I do not want to use too much media for the resuspension, can I just resuspend the cells in, say, 5mL media and add 2mL of this suspension into 10mL?
Hi Mathangi
Your procedure is right in a way. But if you want to follow standard protocol in cell culture laboratory, you have to trypsinze your cells in T-25 flask. Then suspend the cells in 1 ml of the medium and take a cell count using hemocytometer. By taking a count you will have an idea about the health status of the cells in terms of viability. After this you resuspend three times the cell number in 10 ml complete medium in T -75 flask. I presume you may be having a particular seeding density for your T-25 flask. So it will be three times that number for your T-75 flask.
Thank you.
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