10 replies to this topic

[cellthetruth]

Hi everybody,

i have a problem concerning a viability stain. For my experiments I need to permeabilise the cells over night (for treatment with an agent, not for antibody staining). Unfortunately this is indispensable! As the dyes I know, e.g. PI, work with respect to cell permeability this is not possible for my purposes... my cells would look like dead even though they are not, right? Or am I missing something here?

Does anyone of you know a method to stain permeabilised cells?

Please help, thank you in advance!!!

Best,
Martin

[Iwenim]

Hello Martin,
there is the possibility to stain your cells with EMA (Ethidiummonoazid) before.
EMA is binding covalent on the DNA.
Add EMA to the cells incubate about 10 in the dark on ice and further 20 min of incubation under strong light.
Then wash the cells and go on with your normal treatment.

Furthermore there are Live/dead cell staining kits from companies, using different dyes than EMA.

Hope that helps.
Iwenim

[cellthetruth]

Hi Iwenim,
thank you for your quick reply.

As far as I understood (from Invitrogen Product Info-Sheet) EMA also works with cells that have compromised membranes. But that is exactly my problem! I treat my cells for 24 hours before analysing with permeabilising agents and then like to do intracellular staining, i.e. the cells are more or less constantly permeable until I load the samples. Instead I am looking for something like AlamarBlue (which I can not use in my assay).

Any suggestions/experience?

Thanks,
Martin

[bob1]

Could you live-dead stain before permeabilizing?  I think PI would work for this, though I dont know how much of it would wash out with subsequent incubations.

[leelee]

I may be way off the mark here, but when you take away the permeabilisation agent, won't the cells recover? I don't know how long this takes, but you could maybe leave them to recover for a while (in normal media or whatever) and then use a standard live/dead dye?

(of course, that won't work if you have to fix your cells whilst permeable)

[cellthetruth]

Thank you for your replies!

@bob1: Something like this was my first guess as well. But a treatment with the permeabilising agent will subsequently kill some more cells then which would result in false positives. At least this is what I would expect.

@leelee: would be nice. but actually I like to monitor my cells under influence of stress agents. therefore it would falsify my results when i give them time to recover...

Most probably I will try to set up a standard by staining with conventional dye and then gate FSC/SSC.
As leelee said, staining after cells recovered, these compared to untreated cells, could give me an idea of how the cells "like" the permeabilisation...  This should result in an approximate area where I can expect my cells to be alive.

Thank you all for reading & replying to my posts!
Good luck with your experiments and good luck with the reviewers

Best,
Martin

[bob1]

You could try a mitochondrial uptake dye such as Mitotracker - should only be taken up by functional mitochondria... i.e. live ones.

[MicroBone]

you could try to use fixable viability dye efluor from ebioscience.
here you have link http://www.ebioscien...-efluor-780.htm

[cellthetruth]

Thank you bob1. Good idea, didn't think about that!
Thank you Microbone. Unfortunately I can not see how this dye would work.

I think i'll stay with the Mitotracker as they are available in various colors.

[ascacioc]

Annexin V also labels the apoptotic cells by binding PS on the surface of the cells. But only the early apoptotic cells... so... I don't know how useful it would be for you. Check it.

[waldomedina]

I use the LIVE/DEAD® Fixable Dead Cell Stain Kits (Invitrogen). It works very good even when you want to fix the cells.
You have to use it before the fixation. The flow cytometry results are the same as the unfixed.