Trypan blue kill cells generally after 5-6 min or more time than you described, maybe your cells are suffering during the dettachment with tripsin. What's about the osmolarity of your trypan blue staining solution?

What about the protocol?

the thing is, i used to count cell after enzymatic dissociation. no problem. after cultured, when I tried to count, that when everything goes wrong. I tested the cells in haemacytometer chamber with and without trypan blue. the cell is okay without trypan blue but was destroyed with it. concentration is similar used after dissociation and cultured with media. I suspect it was the media-related issue. IS there any salts that interfere with trypan blue to the cells? the protocol is similar to the standard protocol. the cells may also be dying. its a primary cells. thank you for reply.

Oh, and I used pre-made Trypan blue from Sigma.

I think because the cell line you are using is of primary culture, the cells are dying, and thereby trypan blue is staining the whole. Have you tried trypan blue with secondary cell lines???

The trupan blue solution has to be isotonic, if not, it will disrupt the cells very rapidly. Some people prepare it with DI or distilled water. This gives you a hyotonic solution. Not recommended.

Hmm...I think because my cells were cultured in a very high salts concentration media, thats whay when use trypan blue and caused cell lysis. I'm the only one used it and it just a few days after the i used it again. I will try with another batch and other types of staining after this. Thank you very much

If what you saw was that the cells turned to light-blue color quickly after mixing with trypan blue and loading onto a hemocytometer, I'd think the reason was the residual (not thoroughly rinsed out) alcohol or detergent used to clean the hemocytometer. Some other times we also obserevd such light blue color when cells were fragile or weak, such as when they just came out of thaw. I assume your statement (that cells were destroyed by trypan blue quickly) meant the cells mostly turned to blue color, light or deep. I also trust you did not always observe this(?). Good luck.

Thanks for the insight Jinyou, however, when I said the cells was destroyed, it literally mean the cells totally burst and leave a residual clump body. No visible cells is present. I had the same sample, one was treated with TB and the other is not. I saw complete cell shape with non-treated but the treated with TB...only debris clumped in dark blue. And you're right., it's only happen after I cultured the cells in high salt media.

Which buffer do you use to solve trypane blue?

What I assum is that your system is not ISOTONIC. I think you should check this option because Trypane Blue dilluted in water usually lysis cells, particularly blood cells.

Dear Mohammad, I use commercial TB from invitrogen. Don't have any problem when I used it after cells dissociation. Only happen after I cultured the cells in high salt media. Considering the buffer used for dissolving TB, I actually dilute the TB in PBS with the cells. I tried 1:2, 1:5 and 1:10. Will the media component reat with TB, like pH change or specific salts?

So, I don't know about the procedure you follow for counting the cells but in this case, you can collect the cells before staining with TB, then discard the medium and resolve the cell pellets in an ISOTONIC buffer like PBS or HANKS...

Hi, I actually have a question regarding trypan blue staining. Every time I use trypan blue to stain cells, I get some brown staining and I am not sure what that is? Is it just cell debris? Can someone help me find out what that is?

Dear Mohammad, yes, resuspend the cells in isotonic buffer is good, but have to leave them for few min before subjecting to TB staining.

Dear Zana, does the brown staining on the cells or unknown particle? Is it old staining or a new one? Do you use a cell line or mix cells like mine. In my case, I had mixed cells and weird color can come from all sort of thing especially if the cells dissociated from pigmented skin tissue.

Try diluting the Tripan Blue in Ringer's solution.

I find Ringer's solution better than PBS for cell viability and counting. PBS is a tampon sollution, whereas Ringer's solution has glicose wich helps through counting

Maybe you can try to dilute even with the serum-free medium, i.e. The same that you use for cultures, specially if without phenol red..I have never done but I think it works and you are sure that the solution is isotonic

Adding serum reduces the toxicity of trypan blue. Take a look this paper: https://www.researchgate.net/publication/282121055_Application_of_a_non-hazardous_vital_dye_for_cell_counting_with_automated_cell_counters

Article Application of a non-hazardous vital dye for cell counting w...

Trypan blue is cytotoxic. What final concentration are you using? You should be using a final concentration of 0.1%  Often Trypan blue is sold at 0.4%.

Dear everyone, thanks for the advises, insights and paper. All the discussion had helped me finish my PhD. I still cannot point out what happen to the cells when TB was added, but my closest bet would be due to the salts incorporation. At the end of my work, I resort to fluorescence to count cell and determine the cell viability, which was more practical when having a lot of replicates in the experiments. 

Still, it will be very interesting if anyone able to explain TB interaction with the cells at high concentration medium.

Could anyone tell me if a Trypan Blue stain can be performed on Primary cells in DMEM (medium contains phenol red). Thanks

Trypan Blue got its name from the fact that it kills Trypanosomes. How does it kill cells? What is the mechanism of dye exclusion by live cells?