Hi everyone
I am trying to perform live dead assay in a mammalian cell line. I am using DAPi and PI, i.e. ideally DAPI staind nuclear material in all cells and PI must stain nuclear material of cells whose membranes have been compromised i.e dead cells. Howeevr, I am getting complete staining from both the dyes in all the cells. What could be going wrong? Dye concentraation? no of washings? staining protocol?
Plz help
Live cells have intact membranes hence able to exclude a variety of dyes that easily penetrate the damaged, permeable membranes of non-viable cells. Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm. Because of these spectral characteristics, PI can be used in combination with other fluorochromes excited at 488 nm such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE).
Hi Shreyasi,
Sorry for asking a rather stupid question (but sometimes they quickly lead to an answer). I don't know how you're treating / staining your cells. Is there a possibility that your cells die during the procedure?
@Angela..cells are grown on coverslips and trated for 24 hours, following which, media is removed, cells are washed with PBS and then fixed with formalin. After this stained with DAPI and PI. After staning cells are washed with PBS and visulaized. Yes I am afraid that cells that die during the treatment may get detatched from the coverslip, but I dont what is the solution for this and why live cells also take up PI
The order of your protocol might be your problem. The fixation essentially wrecks your cells and snaps them at their current state (but dead). Afterwards, you cannot distinguish dead and alive cells anymore by PI (to my knowledge). I'm not entirely sure , whether it'd work to do it the other way around:
First you treat, then wash, then stain with PI and DAPI and then fix at the end of it. You could try both and compare (so once washing, staining and fixing, and once just washing and staining). You would then of course have to analyse your samples asap.
When doing fixation after DAPI and PI staining (as recommended above) I would recommend to wash your cells multiple times (with large volume of buffer and high concentration of protein =( 10% serum), then buffer alone with minimum 1-2% of protein) to "neutralize" staining before fixation as DAPI and PI tend to leak from dead cells into solution and may stain "de novo dead" (after fixation) cells, if they are not washed well enough. But from practice I would not recommend to do this kind of staining. For dead/apoptotic stain is better to use Annexin V.
There are also Fixable viability amine dyes available like Aqua dead, Zombie, etc. that brightly stain cells that were already "dead" before fixation, and much less stain cells that "died after fixation". This dyes bind amines, and are recommended for flow cytometry, but there are some that can be used for fluorescent microscopy as well, depending on filters availability.
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