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protocol for Trypan Blue exclusion assay - How to do trypan blue exclusiona ssay on adherant cells? (Nov/06/2007 )


I want to find out cell viability using trypan blue exclusion assay. I transfect my cells(HCT 116 cells) with anti-miRNAs using nucleofection technology. 7 hours later, I want to look at the cell viability.Since HCT116 are adherant cells, should I trypsinize them and then do the cells viability assay? Also, do we also take into account the floating cells? It would really hlep if anybody could give me a protocol to do this assay on adherant cells. (along with the calculations). I can see protocols for cells whihc are not adherant, but could not find one for adherant cells.

Thank you in advance.


why do you want to measure your cell viability by trypan blue its not an accurate methode?
and second i know it need a hemocytometer to check the blue cells so it need suspension cells or trypsnization of your adherent cells.


Hello maggie,

I work also with adherent cell. When I want to realise a trypan blue assay, I just need to detach my cell with PBS-EDTA, Trypsin or scrapping (depending of your cell of course). After detachment I take for example 50µl of my homogene solution of cell and add 50µl of Trypan blue. Wait 2' and count the white and blue cell with a thoma cell. It's exactly the same protocole for suspension cell excepted the detachment.

Hope to help



Outline of the protocol I use:

Detatch cells for passaging
After resuspending, take 10µL of the suspension and add to 90µl of Trypan blue. Mix well with pipette, then place 10µl of the Trypan blue suspension on the Hemocytometer, and count the cells in the four 4x4 boxes. Divide that number by 4, then multiply out the proper dilution factors, and you get a count in cells/mL.

Hope this helps!