Hello, this is my first post. I was wondering if anyone here as any experience working with lymphocytes extracted from blood and checking their viability with trypan blue exclusion. I get the impression that I am overcounting dead cells or that I may be counting red blood cells that are not fully removed after purification.

 

What are your thoughts on the matter? Thank you.

trypan blue is used as an cell exclusion method and thus when the cell membranes are intact then this should in principle exclude trypan blue due to selective pressures which is not seen in dead cells and thus the cells appear blueish which is lighter than the surrounding but darker than the background. With red blood cells, the cells themselves contain pigment and thus in trypanm blue appear dark purple to blue depending, also they are quite easy to recognise. using the haemocytometer should give a rough estimate and always start dilutions 1:10 first and then further until you count less than a 100. Alll samples should be throroughly mixed this also ensures that if an error in counting is encountered then this is across the board and thus takes variability out of the equation!

hope this helps

I am not sure what is meant by

thus when the cell membranes are intact then this should in principle exclude trypan blue due to selective pressures which is not seen in dead cells

.

I guess just poor wording, but I still want to point that the the dye do not associate themselves with living cells due to their (negative) charge.

Now, the question is why do you think that you are overcounting? Is the background too high (trypan blue tends also to stain the serum a bit)? Or maybe do you need a higher dilution?