Alizarin red/Trypan blue stain - (Nov/12/2005 )

For my final thesis I investigated the cell death on porcine corneas before they could be transplanted. One of the basic tools for a cell count is a staining of the endothelial cells with Alizarin Red S complex and counterstaining with Trypan blue.
Cellular spaces were colored red, whereas ALL cells were slightly blue with visible nuclei.

Now my question: how come these cells take up the Trypan blue??? All membranes were intact, the cells were definitely viable.

(Alizarin red: 1 %, pH 4.2, 2 min. Trypan blue: 0.25%, 30 sec.)


Hope you can help.

I worked with trypan blue, but I'm not an expert in this field...
Have you checked the exposure time to trypan blue???
Maybe if you leave your cells in contact for too long, there can be a trypan uptake even if your cells are right...?...
If I'm not wrong, I remember that trypan is also slightly cytotoxic...could it "harm" your cells during the staining time???
Unfortunately, these are just suggestions, so I can be totally wrong...
Anyway I hope this could help...

Thanks for your answer, I have already considered cytotoxic effects, exposure time etc., still there is nothing "wrong" with the cells.

This staining is done in eye banks prior to transplantation on a routine basis to evaluate morphology, but since I come from "regular" cell culture I just don't get it...

I think it probably has to do with the timing thing...

What I remember about trypan blue is: when counting cells and using trypan blue to stain "dead" cells, they just take up the trypan blue first, all the cells will eventually take it up and that is why you only wait 1-2 min between adding trypan and doing viability counts...

So I think that may be why all the cells stained...