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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!

-kimj-

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.

-jerryshelly1-

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.

-Tabaluga-

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!

-kimj-

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... :(

-Tabaluga-