Hello. So I had seeded some 48-well plates with 70,000 cells/well (human osteoprogenitor) on a calcium phosphate substrate scaffold.
After 24 hours I wanted to study the overall attachment so basically I incubated I removed the scaffolds from the wells and placed in to a new well plate.
I washed with PBS and then incubated in a trypsin 1x solution for 30 minutes to allow for the cells to deattach from the scaffold.
I then neutralized with media and centrifuged the solution to form the smallest dot sized pellet.
I removed the solution and reconstituted in 1 mL of media and then using 10 uL of the solution, mixed it with 10uL of trypan blue.
Transferred 10 uL to side A and side B of a chambress counting slide and used the corresponding automated cell counter by life technologies to count the cells.
I somehow got a bigger number - for example 4.5x10^5 cells alive/ mL
The time point was only 24 hours so it’s not possible that the cells divided that quickly and multiplex in that number so fast.
Can someone please help me understand the principles behind automated cell counting because I believe the machine maybe possibly multiplying by a factor to estimate the number of cells? Please help because clearly the machine won’t count cells which aren’t there, I just don’t understand why it’s spitting out such larger numbers.
Please it’s my last experiment of my thesis and I just need a little help please.
Dear researcher
See the following papers method
Cell viability analysis using trypan blue: manual and automated methods
Here are some easy videos to answer your questions:
Hope that helps you
https://www.youtube.com/watch?v=wPeNK0pRIpA
https://www.youtube.com/watch?v=qfT9uqqme8c
https://www.youtube.com/watch?v=Z9-PSSWK9N0
Dear Ghosh,
please make clear that the sample is mixed.
If you use a Cellcountingmachine, only use fresh matierial. Plastic slides can expire and the Machine will count the plastikparts as cells.
If you are working with differentiated MSCs a lot you should check-out Cellseeker https://www.cellseeker.org/). This is an easy and free to use app to organize and inventory your cell collection online.
For attachmen test, i carry out with SEM test.i have not experience in this feild.