Cunterstaining Trypan blue - (Feb/19/2009 )

I am meaguring of cytotoxicity using cell culture in 24 well plate and staining dead cells directly on the plate with Trypan blue. Trypan blue makes the nuclei of damaged cells dark blue. I wonder if there is a way to stain all the cells including live cells with a different color to help better visualisation of all cells? That would especially help for better photographs and image analysis.
I tried Eosin staining and it seems to me that ethanol in Eosin stain is washing away the Trypan Blue color. Any ideas?

How is this an accurate measure of cytotoxicity? Dead cells lift off the plates and float around in the medium. I personally would collect the medium and lift the attached cells off and combine the two, then do a conventional haemocytometer count.

Trypan blue should not just stain the nuclei, it should stain the cytoplasm as well, as cell membrane permeability increases when the cells start dying.

maxrempel on Feb 19 2009, 04:07 PM said:

There is a live/dead stain (fluorescent-based) available from Invitrogen. Below is the website for the stain....check it out...maybe it is applicable for what you are trying to do.

here is the link: http://www.invitrogen.com/site/us/en/home/...Brand-Page.html

Cheers!

Wolverena on Feb 19 2009, 08:30 PM said:

Hi There,

Have you tried using Promega's Cell Titer Glo Cell Viability assay kit. I have been experiencing problems and see variable results each time. I plate 5000 cells/ well in a 96-well plate. Have you been able to get consistent results using the invitrogen live/ dead stain?

Thanks

Pmaj

Wolverena on Feb 19 2009, 08:30 PM said:

Hi There,

I looked up the invitrogen website for the live/ dead stain. They have a couple of those for mammalian cells. The LIVE/DEAD® Cell Vitality Assay Kit *C12-resazurin/SYTOX® Green* *1000 assay has the same dye (REsazurin) as in the Promega Cell Titer Blue kit. I have not had much luck using this particular Promega assay kit for cell viability.

Invitrogen has 4 different kits which use 4 different dyes, could you please specify which one worked best for you.

Thanks

Pmaj

pmaj on Mar 6 2009, 11:01 AM said:

Hi Pmaj

Unfortunately, I don't have any experience working with mammalian cells.
I have used the live/dead staining from invitrogen for bacteria, which worked well. The dye in bacterial kit is SYTO® 9 dye propidium iodide.
I would suggest that you call technical support and explain your problem. They might be able to help you troubleshoot and suggest the right kit. And try to contact the Invitrogen sales Rep from your area....the Rep might be able to give you a sample/trial kit to test the staining on your cells. Good Luck!

Wolverena on Mar 6 2009, 01:56 PM said:

Thanks a bunch Wolverena ,

a free sampe would be neat to try out first

Pmaj