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...
I am having the same problems with my PBMCs. I am losing about 30-40% of my cells after incubating them for 1 hr at 37C. When I say "lose" I literally mean there are less cells to count on the slide. It is not a viability issue. At first I thought it was becuase I was using a flask and the monocytes were adhering to the flask, but when I tried using a 50mL conical tube, the same thing happened. Any advice or insight would be greatly appreciated!
I have experience in culturing cells that clump together in suspension. What you see is that, after trypsinization and resuspension in medium, the cells will stick together and form clumps the longer they're left in suspension on the bench. This can cause the cell count to go down to less cells per ml. In our lab, we use a Counter that also shows a graph of size vs amount, so I can make a discrimination between counted particles that are too small or too big and thus are suspected to be something other than cells. If the cells clump together, a shift in the graph to more large particles counted per ml.
If this is the problem, then the solution is pretty easy: try pipetting your suspension up an down again before counting the second time. Or you could try looking at the cells under the microscope. Transfer the cells from the tube back into a T25 and have a look.
To further elaborate on the cell loss I am seeing with my PBMCs; we are using frozen cells. After thawing allowing to incubate for 1hr, we did see clumping of the cells. This would naturally lower our cell count as the clumps of cells are being excluded and we are not counting a homogenized cell suspension.
That being said we used DNAse to prevent clupming which worked very well. The DNA strands were acting as a "binding agent" causing a gooey clump of cells.
Unfortunately, even with elimantation of clumps, we still have the problem of losing cells. Again we are not losing cells due to viability issues, there is no increase of cells taking up the trypan blue stain. Furthermore, I am not seeing an increase of debris either so I don't think it's an issue of the cells lysing into tiny particles.
Could 30-40% of my cells really be sticking to the 50mL conical tube???