The cells I'm working with (BY2 and NT1) are clumpy and they don't leave each other; for using hemocytometer many times they can't pass through the cover slip so that many stay just behind it. Trypsinization with EDTA didn't help. I think this problem can be seen even when I need to use a pipette with a small tip for getting 10 microliter of them as they can't go in the tip. So, sometimes I cut the tip for taking them.
I was wondering whether or not hemocytometer can be in different sizes to have a deeper area underneath for letting to cells to go inside the grids?
Regards,
Have a look at your clumps of cells in a dish. I imagine you won't be able to count individual cells. So a deeper hemocytometer is unlikely to help.
If you need single cells, then enzymatically harvesting after a shorter time in culture usually helps. This is because they don't have as much time to generate massive amounts of extra cellular matrix. Also try other enzymes: trypsin, collagenase, accutase et al.
If that is not possible you might try counting nuclei with fluorescent dyes such as Hoechst 33258. Or extracting and quantifying DNA spectrophotometrically.
I'm confident that if you can't get the clumps into a hemocytometer, you'll not be able to resolve and count individual cells within the clumps. The solution is in getting a better suspension. Try other enzymes (accutase, collagense etc) and conditions (warm, RT: longer, shorter). Maybe cell lysis is clumping them together. DNase sometimes helps.
But usually the problem is that the cells have been left too long between passaging. Too long and some cells make nearly impenetrable extra-cellular matrix. Shorten the passage time and adjust the seeding density.
Alternatively, use Hoechst 33258 to visulise and count nuclei.
Alternative alternatively, lyse a sample of the cells and measure DNA spectrometrically or fluorometrically.
Hi Dear Lyons,
Thank you so much for your response. you are right the i can't see the individual cells. maybe other enzymes can help, i mean i hope they help otherwise i am in a big trouble. i'll try those enzymes.
Cheers,
Hello
Agreed with the research, you must make sure that cells non-conglomerate on each other
To answer your question on the size, yes, there are different sizes of hemocytometer you can visit the following link
http://www.hemocytometer.org/2013/04/11/hemocytometer-square-size/
I wish help you
Best wishes
Salam,
Thank you Dear Saleh. So, seemingly the grid has different size of chambers.
Dear Researchers, By2 / NT1 cells are so clumpy even when I used the cells in low concentaration, for example in the first day of passaging. Accutase help the cells to be seen more clearly but doesn't make them separated. I can't see the individual cells. EDTA couldn't do either. But when I used it in the higher concentration, it caused the high mortality of the cells. I also understood that in the less concentrations it changes the cells morphology. However, according to this result by now, I think using hemocytometer is not possible for them. Nevertheless, I am wondering how a lot of papers referred to hemocytometer. So, a question remains to be answered that why they were able to count by hemocytometer but I am not?
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