I used several methods for breaking them from each other but none of them worked. For drawing growth curve I need to be able to count them every 13h,but the way they are together makes it hard for me to count them using hemocytometer.Even enzymes like trypsin or physical shakes such as vortexing were not able to break them apart, which possibly can effect their viability too.
Do you think that fluorescent microscope and using PI and FDA is the only way? it takes time and I don't prefer it since I should repeat this work a lot if any suggestion for hemocytometer you have please let me know.
thanks,
Dear Mahbobeh,
To develop growth curve, you can use different parameters. You can take fresh and dry weight measurements in a given volume in replicated experiment. You can also use packed cell volume as an estimate of growth. Fresh weight increases are well correlated to the growth of BY-2 cells, If you are interested in understanding the speed of cell cycle, you can stop the cell cycle using blocking agents such as diphenyl urea, aphidicolin, release and estimate the doubling time which is equal to the cell cycle in hours.
If you are interested in microscopic measurements, it is tedious to count, so I would take microphotographs at the same magnification using same dilution, and then count them leisurely. Yes FDA would be a good stain if you want to count only living cells. For live/dead ratio you can stain with FDA and Evan's blue and take microphotographs in light and fluorescent settings.
I have attached an article that may be useful
Hope this helps.
Hello,
I always try putting 40 µL of cells added to 40µL of trypan blue between slide and cover glass. And observe under microscope on 10x and count with a double counting device (fisher 02-670-12,Bal Supply Llc,No.: 202C).
FDA and PI staining could give you a general answer for your count but I don't believe that the signal of Pi is specific at the cellular level, sometimes it colors cell wall or contour my cells even if thr alive. Although it works like magic with roots and whole organs, I prefer other staining methods over FDA/Pi.
Trypa blue : Usually I take my cells in the third day after transfer. But if you see that the density is high, try counting on the second day maybe? Or even do some trial with volume, like reducing the volume of the transferred cells (maybe 4ml to 50 ml) compared to the fresh medium: than compare that with cells that you transferred in a normal ration (6ml to 50 ml). Hope my answer helps! Good luck :)
You could try using a MTT assay to create a growth curve http://web.mnstate.edu/provost/mtt%20proliferation%20assay%20protocol.pdf. There of course would be some issues with clumping preventing some cells from fully participating in the assay but it should be able to distinguish treatments that significantly affect the growth rate of the cells.
If the clumping is due to bits of RNA or DNA released from dying cells, you could try RNAase/DNAase incubation after trypsin digestion. If you think its actual cell-cell ahesions, like cadherin junctions, you could try adding EDTA to the trypsin buffer to chelate out divalent cations essential to the formation of tight junctions.
Or, you could try an enzyme solution such as Accutase, with which I've been able to digest clumped cell colonies into single-cell suspensions, with no apparent cytotoxicity even after 30min of incubation. Or, use a soluble formazin dye, to get relative cell numbers, similar to what Daniel suggested above.
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