cell counting

[cellchem]

hello, all

I am trying to count the cells (hemocytometer) using Trypan Blue. However I have difficulty differentiating dead cells from live ones (the cells all looked same under microscopy). I am afraid I did some thing wrong (stain time? microscopy setting? ...) .

Can anyone show me a pic of cells stained w/o Trypan Blue so that I can have an idea what the cells should look like when using this method? Thank you!

[July2009]

Hi Cellchem,
For our hemacytometer (approximately 20µl/chamber) we use 2µl trypan blue for 18µl of cell suspension and we can see dead cells coloured in blue.
Hope this helps

[wr_1a]

thanks

[rdittmar]

When using trypan blue, the bright cells (blu-ish tint) are live cells. The dark cells (dark blue splotchy circles) are dead.

[Zagami]

Viable cells as dye exclusion method : trypan blue vital stain is a simple way to evaluate cell membrane integrity that is not absorbed by healthy viable cells. When cells are damaged or dead, trypan blue can enter the cell allowing dead cells appear blue to be counted. In my experience “F. Zagami (1992) : Reduced combined immunologic activity in case of centroblastic-centrocytic lymphoma. Con. Med. 1, 1-11, in which were used Trypan viability dye as following recommended in specific derived kit (Fagochemical) : dissolve 0.4 g Trypan blue (Sigma cod. T0776) in 95 ml dH2O containing 0.81 g NaCl, 0.06 g KH2PO4, and 0.05 g methyl-phydroxybenzoate. The solution is heated to boiling to dissolve all materials, cooled, and adjusted to pH 7.2 with 1 N NaOH. The final volume was then adiusted to 100 ml with dH2O. The dye solution is sterile-filtered and stored at room temperature. Procedure : Maintaining a ratio dye:cell suspension between 1:5 and 1:10, in relationship to cellular density of the suspension if < to 1.5x10^3 or > to 1.5x10^6 respectively, to distribute in test-tube the cellular suspension and then the dye. Mix gently and incubate for 5 min. at room temperature. Examine one drop, placed on clear glass slide under coverslip, under phase-contrast optics, and the number of cells is counted in previously marked fields. Cells that appeared as dark blue on a lighter background are counted as nonviable.