What if your seeding in a T75 flask or in a cell culture disk? The T72 can take a volume of 20ml and the dish 10ml, do I use those volumes to calculate cells needed?
It does not matter whether you are seeding in a T75 flask or in 10 cm plates, the most important thing you should care about is the cell number you need. First make sure how many cells you need, and how many plates you are going to seed. Then, calculate the total number of cells you need, [for eg, 1x10^6 cells for 3 T75 flask= 3x 1x10^6 cells ] and divide it by the cell concentration you got from cell counting. And then get that amount of cell suspension into 15 or 50-mL tubes depending on volumes and centrifuge, remove supernatant media and resuspend cells in your desired volume of fresh media and seed them in the plates as you calculated before. The final volume of media doesn't have any relation with the initial cell number counting and calculation but the media volume should be enough for the cells you seeded in the plates. I hope this helps, Thank you
Thanks for the information but I want to know that after centrifugation of 8.945 mL suspension, in how much fresh media I will do resuspension and how much from that i will transfer to each well of 12 well plate?
We calculated for n+1 that mean 12+1=13 , in 12 well plate you can add 1 mL in one well. Therefore you can add 13 mL fresh media to resuspension the cell and canadd 1 mL to each well.
I just took 12 mL for the round figure, because I use a multichannel pipette to seed the cells in 96 wells. If I took a lower volume, then I could not get enough volume at the last column of my 96-well plate. The idea behind taking higher volume is to make sure that you have enough volume while seeding cells. If you feel comfortable with taking 10 mL, then you can calculate for 10 mL, which is okay if you use a single-channel pipette to seed the cells.
It does not matter whether you are seeding in a T75 flask or in any well plates, the most important thing you should care about is the cell number you need. First make sure how many cells you need, and how many plates you are going to seed. Then, calculate the total number of cells you need, and divide it by the cell concentration you got from cell counting. And then get that amount of cell suspension into a 15 or 50-mL tube and centrifuge, remove supernatant media and resuspend cells in your desired volume of fresh media and seed them in the plates as you calculated before. The final volume of media doesn't have any relation with the initial cell number counting and calculation but the media volume should be enough for the cells you seeded in the plates. I hope this helps, Thank you
Thank you very much for such a nice video i was looking for thi calculations as a new master student in an immunology lab....i need to contact you for some questions if you do not mind kindlt provide me any means of contact of you,any phone number any email any research gate account because i desperatly need some help regarding calculations for my experiments.....i already subscribed your channel.. Thanks again. i am waiting for your kind response.
Great video thank you! I just did not understand why you added n+1 and n+20 "for sufficient volume"? What does this mean?
+1 or +20 is just a margin of error having a margin of error ensures that you have enough samples for your wells.
What if your seeding in a T75 flask or in a cell culture disk? The T72 can take a volume of 20ml and the dish 10ml, do I use those volumes to calculate cells needed?
It does not matter whether you are seeding in a T75 flask or in 10 cm plates, the most important thing you should care about is the cell number you need. First make sure how many cells you need, and how many plates you are going to seed. Then, calculate the total number of cells you need, [for eg, 1x10^6 cells for 3 T75 flask= 3x 1x10^6 cells ] and divide it by the cell concentration you got from cell counting. And then get that amount of cell suspension into 15 or 50-mL tubes depending on volumes and centrifuge, remove supernatant media and resuspend cells in your desired volume of fresh media and seed them in the plates as you calculated before. The final volume of media doesn't have any relation with the initial cell number counting and calculation but the media volume should be enough for the cells you seeded in the plates.
I hope this helps,
Thank you
Thanks for the information but I want to know that after centrifugation of 8.945 mL suspension, in how much fresh media I will do resuspension and how much from that i will transfer to each well of 12 well plate?
We calculated for n+1 that mean 12+1=13 ,
in 12 well plate you can add 1 mL in one well. Therefore you can add 13 mL fresh media to resuspension the cell and canadd 1 mL to each well.
howd you figure out that you had to add 12 mL of media for the 96 well plate example
I just took 12 mL for the round figure, because I use a multichannel pipette to seed the cells in 96 wells. If I took a lower volume, then I could not get enough volume at the last column of my 96-well plate. The idea behind taking higher volume is to make sure that you have enough volume while seeding cells. If you feel comfortable with taking 10 mL, then you can calculate for 10 mL, which is okay if you use a single-channel pipette to seed the cells.
great video. was wondering if i would like to cite this calculation method. who should i cite??
Hi, Thank you for your compliment. You can cite my paper here.: www.mdpi.com/1999-4923/14/1/157
Thanks for the video . how did you come up with 120 if you use n+20. I thought n in this case is 96 so if I add 20 to that it will give me 116.
rounded up to 100 to make calculations easier, 100+20=120
@@joshzussman1289 i know this was 2 years ago but where ever you are thank you it was driving me crazy
What to do in case of T25 flask?
It does not matter whether you are seeding in a T75 flask or in any well plates, the most important thing you should care about is the cell number you need. First make sure how many cells you need, and how many plates you are going to seed. Then, calculate the total number of cells you need, and divide it by the cell concentration you got from cell counting. And then get that amount of cell suspension into a 15 or 50-mL tube and centrifuge, remove supernatant media and resuspend cells in your desired volume of fresh media and seed them in the plates as you calculated before. The final volume of media doesn't have any relation with the initial cell number counting and calculation but the media volume should be enough for the cells you seeded in the plates.
I hope this helps,
Thank you
@@bio-capsule5885 Thank you for this explanation. It really helped!
Thank you very much for such a nice video i was looking for thi calculations as a new master student in an immunology lab....i need to contact you for some questions if you do not mind kindlt provide me any means of contact of you,any phone number any email any research gate account because i desperatly need some help regarding calculations for my experiments.....i already subscribed your channel.. Thanks again. i am waiting for your kind response.
jshrestha@mgh.harvard.edu