I'm conducting a Puromycin dihydrochloride antibiotic selection in CHO cells, according to several protocols, the medium is replaced with fresh medium containing Puromycin every 48 hrs, in order to generate a kill curve, they only count the cells on the day-10 of the experiment by MTT assay or manual cell counting using Tryphan blue. I was wondering is it necessary to count the dead cells during the medium replacement (every 48 hrs)? and if it so, do you typsinize the cells (in the middle of the experiment), or do you just manually detach it by pipetting up and down because you only need to count the dead cells every 48 hrs? can anyone give me some insights? thank you.
Hello
You can check for cell death every day by observing the cells under the microscope as dead cells are not bright. Counting the dead cells after every 48 hours is not recommended. But if you still would want to do so, you may remove the spent medium from the culture and spin it down at 200-300 g for 5 minutes to obtain a pellet of dead cells. It would be difficult to count dead cells because these cells would form cell debris and most likely would give an inaccurate count.
You should not trypsinize the cells or pipette up and down because dead cells often round up and get detached from the substratum. This action of trypsinization or pipetting up and down would cause unnecessary interference in the assay.
There is no need to count dead cells after every 48 hours. Please follow the protocol.
I hope this helps.
Best.
Hi,
Reading the description, It is not required to trypsinze cells and if its not in the protocol, should not deviate from it and detach the cells. You can directly collect and count the cells just after washing. If your cells are dead then they would be washed away. So no need to using trypsin. End of 10 days you can run an MTT to see the amount of live cells.
I would personally count the dead cells. As you are culturing for 10days, there would be increase in number of cells so it would be wise to count the dead cells.
I start the kill curve after the transfection for puro, G418, MTX, ... selection. I inoculate cells into 6-well (2mL/well) at 5E5 cells/mL. The next day, i start to add the selection with different concentration, from lowest to highest. Count on Day 3, Day 7, and Day 10 using trypan blue 30uL/time. Based on cell count, the viability, i made a graph to optimize the the concentration of selection when i start to step wise increase as well as the max concentration i can increase up to. You don't want to start the selection at too low, or too high concentration.
thanks a lot for the insights, everyone Malcolm Nobre Kirankumar Gudagudi | may I know, when you do the trypan blue cell counting on the day 3, day 7 and day 10, do you trypsinize your cells? Dai Quach
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