Live/Dead Staining of already Permeable CellsLive Dead Stain Cell
Posted 12 September 2012 - 01:41 AM
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!!!
Posted 12 September 2012 - 02:07 AM
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.
Posted 12 September 2012 - 02:31 AM
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).
Posted 12 September 2012 - 01:40 PM
Posted 12 September 2012 - 06:18 PM
(of course, that won't work if you have to fix your cells whilst permeable)
Posted 17 September 2012 - 04:37 AM
@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
Posted 17 September 2012 - 12:57 PM
Posted 18 September 2012 - 07:52 AM
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.
Posted 19 September 2012 - 02:19 PM
Posted 06 February 2013 - 10:35 AM
You have to use it before the fixation. The flow cytometry results are the same as the unfixed.