Hi,

I am looking for a viability marker to stain alive white blood cells and still working after fixation.

Here is my workflow for you to better understand (and tell me if my asumptions are wrong).

1) I am studying white blood cells from total blood (EDTA tube).

The next step must be perfurm in the hour after the sampling.

2) I take a certain volume of blood and had a buffer with fixative agent during 8 min (blood is diluted in 1:1).

3) Then, I make the blood go through a filter to stick fixed cells on it.

4) Finally, I stain the filter with May-Grünwald Giemsa - MGG (resulting in colors from pink to blue with purple) to see the nucleus, cytoplasm et identify my cells under microscope (brightfield).

My idea is to be sure that my cells I identify by MGG were well alive before the 8min fixation.

--> So, I would love to have a viability markers that I could identify in brightfield (so more a dye?), in different colors other than pink/blue/purple and that is not toxic for cells (no apoptosis or death process). If you have an idea.. let me know please :)!

I have made some research and here I found :

• metabolic stainer such as XTT could have worked but it need an incubation of 2h at 37°C which I can not dot.
• I found the neutral red but I am not sure if incubation needs to be 1h too or not? if there anyone who has experienced neutral red to stain alive human cells?
• trypan blue : I have read that it could go through all the cells if time is not short and I am not sure it is still working on fixed cells. I also read it is toxic for cells (but how long?) We could think of 2 min of incubation with trypan blue before the 8min fixation but I do not know if it is a good idea as I can not have a washing step between blue trypan andfixation (so I suppose all my cells will be trypan blue because of the fixation that can make small cells go into the cells?) and also because of the toxicity.. if someone have already test this?
• finally, I could try fluorescent dye (blue, Cy2 or Cy5). I though about DAPI at low concentration, 5 min before the 8minfixation. But I means I have to use a microscope with both brightfield and fluo which means the identification of cells will take more time. But If I have not the choice..

Thank you for you returns! :)