I'm carrying out a FACS assay using intracellular markers, for which I am using fixed cells (2% PFA) and permeabilized with Triton X.
I'm having trouble identifying and gating out debris in my sample, which as I understand are the events with low FSC and low SSC, so at the bottom left corner of the dot plot.
My question is if adding DAPI to my sample will help me better identify the debris. Will DAPI only bind to the actual cells in the sample, or will it for some weird reason also clump with or bind to the debris?
I'm aware that DAPI binds DNA, so in theory, it should only mark cells in my sample, but something makes me wonder if it wouldn't also stick to the debris?
Thank you.
Hey Gael,
in NON-fixed cells I use DAPI to mark dead cells (similar to PI or 7-AAD), as dead cells are only permeable for DAPI but not live cells. If you fixed your cells and add DAPI afterwards, you will label cells that were dead and alive, so not the best way to discriminate between the cells.
What you can do is label your cells with a fixable viability dye (e.g. this one: http://eu.ebioscience.com/fixable-viability-dye-efluor-450.htm) a few minutes BEFORE you fix them with PFA. Then you can just perform the fixation and staining. Like this you can gate on cells that are negative for the viability dye and exclude dead cells and debris.
I hope this helps you,
Simon
Hi Gael
I agree with Simon. It's always important to have a viability dye, specially because dead cells exhibit an increased autofluorescence. However, everytime you use such dye you must do it before permeabilization. I do it during my extracelular staining, than I wash cells to remove excess dye and fix/perm.
Another importat aspect you have to check is if you cytometer is capable to detect DAPI since you need a specific configuration. You should verify if your cytometer has a Violet laser (405nm) and the 450/40 filter.
Thank you Simon and Andre for your answers.
However, it's still not clear to me if debris will also bind DAPI if added post fix/perm. I am aware that DAPI, when added before fixation, will only label dead cells.
But my question is a bit more technical: If I add DAPI after fix/perm, will it also bind to debris randomly for some reason?
(I understand that adding DAPI at this point will cause all cells to be labelled, whether they were dead or alive before fixing),
Hi Gael
I only used DAPI for fluorescence microscopy so I didn't have cell debris. I know that PI can stain cell debris but I'm not sure if DAPI would also stain them.
Why is it difficult for you to gate out cell debris? Do you have a lot of debris?
Hi, Gael,
I agree with two previous researchers.
Considering the mechanism of how DAPI works, the answer to your question if DAPI only bind to the actual cells would be "no". Because DAPI is able to bind all DNA including mitochondrial DNA if your fixation compromises the integrity of plasma membrane.
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