I need to check cell viability in a flow cytometry assay, and I always use PI. This time because I have different fluorochromes I can't use PI staining so I will use DAPI. But I have no idea about the working concentration. My cells are bone marrow cells and they are not fixed.
And should I dissolve DAPI in my staining buffer or water or PBS?
Yes, PI can be a pain, been using DAPI for >10000 samples now. But beware: it might have a higher permeability in mouse cells! For human PBMC sitting 24h in DAPI solution I found no increased or unspecific staining compared to controls being stained after 24h, but you find people running around claiming DAPI is cell permeable (which is technically true, with a rate being extremely slow, for my cells at least). We excite at 405 and collect at 450/40 or 550/40 on a BC Gallios.
Personally, I stain my PBMC / lymphocytes with a simple "protocol"
i) mAb stain your cells
ii) wash them
iii) resuspend in your final buffer containing 100-1000nM DAPI (my stock is simply in PBS at 100µM and 4°, e.g. dilute stock 1:10 in PBS and add 50µl of dilution to ~450µl of cell suspension which you will acquire yielding 1µM DAPI).
As mentioned you can use much lower concentrations but with a dye being so cheap I find no trouble having a well resolved dead cell population log-wise.
I found that cells stain between 30-45s, so NO need for 15min incubation. But you can check that yourself, just stain irrelevant dead cells by adding DAPI and immediately acquire on fc, then check DAPI channel vs time.
Cheers, just drop a line if you have further questions.
Firs of all a few questions:
1- Do you have a UV laser on the flow cytometer?
2- Are you planning to permeabilize the cells? (to get DAPI into the cells with intact cell membrane very high concentrations are needed and those concentrations are toxic to cells and alter the metabolism of the cells)
yes I do.
I will not permeabilze the cells because I need to know if the cells that I'm using for the flow cytometry experiment are dead or alive. If I have some dead cells they will be DAPI stained otherwise if they are alive they will not be stained by DAPI
So a range between 1/1000-1/2000 will work for you (at least worked for me). One procedure uses 1/5000 as follows:
-Dilute the DAPI stock solution to 3 µM (1/5000 dilution from stock) in staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2). A 1 mL volume will be required for
each cell sample (2 × 10^5 to 1 × 10^6cells).
-Centrifuge the cell suspension (from step 2.6) and discard the supernatant. Tap to loosen the pellet and add 1 mL of DAPI diluted in staining buffer.
-Incubate for 15 minutes at room temperature.
Yes, PI can be a pain, been using DAPI for >10000 samples now. But beware: it might have a higher permeability in mouse cells! For human PBMC sitting 24h in DAPI solution I found no increased or unspecific staining compared to controls being stained after 24h, but you find people running around claiming DAPI is cell permeable (which is technically true, with a rate being extremely slow, for my cells at least). We excite at 405 and collect at 450/40 or 550/40 on a BC Gallios.
Personally, I stain my PBMC / lymphocytes with a simple "protocol"
i) mAb stain your cells
ii) wash them
iii) resuspend in your final buffer containing 100-1000nM DAPI (my stock is simply in PBS at 100µM and 4°, e.g. dilute stock 1:10 in PBS and add 50µl of dilution to ~450µl of cell suspension which you will acquire yielding 1µM DAPI).
As mentioned you can use much lower concentrations but with a dye being so cheap I find no trouble having a well resolved dead cell population log-wise.
I found that cells stain between 30-45s, so NO need for 15min incubation. But you can check that yourself, just stain irrelevant dead cells by adding DAPI and immediately acquire on fc, then check DAPI channel vs time.
Cheers, just drop a line if you have further questions.
Thanks a lot for your so simple protocol, Sander. We are in the same way than you, we are looking for minority proteins so need to used PE labelled antibodies, and PI is a pain, so DAPI is a solution. I will tell you if it works ok here.
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