Cells adhering to walls of conical tube? - (Jun/13/2013 )
I can't explain why the total concentration of my cells reduce by 40-70% over one hour period. Could the cells be adhering to the walls of the conical tube?
I am counting Jurkat, 3T3, HEK and CHO cells in a standard 15ml conical tube (polypropylene) or microfuge tubes. I either pipette triturate 5x minimum or invert the conical slowly 3-5x before counting on our Coulter counter to avoid settling of the cells. Either way, I can start at 7E5 cells/ml, but after an hour the concentration is around 3E5/ml. Could the cells be adhering to the conical wall even after mixing the sample?
It's quite perplexing and any suggestions would be helpful. Thanks!
Have you tried adding your trypsin again before you count the cells a second time? I have never had any problems with cells adhering to falcon tubes.
1. Regarding your hypothesis that the cells adhere to the tube, I once had a problem with cells adhering to walls of small Eppi reaction tubes. The discussion can be found here http://www.protocol-online.org/forums/topic/28080-centrifuged-cells-stick-to-wall-of-reaction-tube/page__hl__+cells%20+sticking#entry147612
2. To your question specifically, do you count viable or total amount of cells ? Because depending on how senstitive the cells are to these conditions, viability may drop within 1 h so there will be a lesser amount of viable cells. I don't know if the Coulter counter automatically distinguishes viable from dead but I think the range of sizes to be measured can be defined in the settings.
Thanks for the input! The link was pretty helpful and confirmed some of the ideas I had as well. But as people have discussed, it would be very time consuming to coat all of the tubes that I use. I don't think I can add trypsin to a sample that is already in another media. To answer your question, Tabuluga, I count total cells, not viable. If the cells were dying rapidly within the hour, I would expect to see more cell debris in the histogram.
Have you tried the more expensive low-binding tubes? I am curious to know how well they work for this situation. Thanks!
Unfortunately I never tried these tubes... in my case I just learned to live with the problem even though I still wonder about it now and then...