I am staining murine skin cells with a variety of fluorophores. When staining with CD45, I only start observing clear separation of positive/negative populations at a 1:12.5 dilution ( 8 microliters of AF700-conjugated CD45 into 100 microliters of staining buffer). I wonder if this dilution is too low (I may be adding too much antibody), and if I may be doing something wrong. I use AttuneNxT flow cytometer. I have already adjusted the voltages. Prior to staining, I digest the dorsal skin in collagenase type 4 and Dnase I. Please see the image. Thank you for your help.
Hi Lazar D. Nesovic,
Can you explain more about the gating strategy you used, as the good separation you reach with 4ug antibody yield more than 50% of cd45+ positive cells from murine skin cells, is this usually expected for digested dorsal skin samples?
usually, different vendors have different recommended dilutions, and if the antibody storage is not appropriate, it might results in weak staining, thus requiring a greater amount to be used. Also, it depends on how many cells you stain, if much higher than 1 million cells it requires a greater amount of antibody.
You can check for previous people who did similar staining for dorsal skin samples to compare how many cd45+ cells you are expected to find, then for the suitable concentration of the antibody you have or replace it.
Have you stained for dead/live cells, dead cells might stain falsely and need to be excluded.
Good luck!
Yassouf
Is it possible to switch to CD45 in APC-R700 rather than AF700? They should be picked up by the same detector, so it shouldn't mess up your panel, but APC-R700 is much brighter than AF700 so it might help.
If you can switch, and you still don't see a good separation, then my guess is that something is up with how you are digesting your samples (it might be stripping off the CD45 - I'm not sure, I've never had much luck with skin leukocytes).
Here's a link to the APC-R700 antibody (I'm not sure if suppliers other than BD have this fluor):
https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/apc-r700-rat-anti-mouse-cd45.565478
Good luck!
-Andrew
Mhd Yousuf Yassouf I excluded the debris from the FSC-H vs SSC-H plot, exclude the doublets in FSC-A vs FSC-H plot, and go straight into the CD45 (RL2-H vs SSC-H) plot. I did not stain with the viability dye.
I stain 200,000 to 250,000 cells in 100 ul of staining buffer. Interestingly, even bright fluorophores such as PE (I stain XCR1 dendritic cells with it) or PE-DAZZLE also require high amounts of antibody to produce clear separation between positive and negative populations.
I shall keep looking at the literature sources. Typically, authors have used AF700, APC/Cy-7, or PerCP/Cy5.5 for staining skin leukocytes.
Thank you for your help!
Andrew Wight I just checked, and APC-R700 fits our flow cytometer's configuration. I will order it and see if I get better results.
For what it may be worth -- after digestion, I perform gradient separation, which leaves me with live cells only, and sends the debris to the bottom of the tube.
Collagenase type IV is not known to cleave off CD45 surface markers.
Thank you for your help!
Lazar D. Nesovic Thanks for the update - hopefully R700 helps, but from looking at your above answers, if you are having trouble with separating PE and PE-Dazzle then it might be a cytometer/setup issue (or possibly something else with your sample prep is making them super autofluorescent?)
When you do instrument setup, do you use comp beads to set compensations? And if so, do you see a very sharp distinction between negative and positive bead populations with the bright fluors? If not, something might be up with your instrument/staining.
Hi Lazar D. Nesovic
If you experience weak staining even with bright fluorophores, It is possible that some cells are stained with the antibodies unspecifically, and weakening your stain of interest, you might consider "FcR Blocking" which block the nonspecific staining (some times it is necessary depending on the sample source, such as high content of macrophages or monocytes ...).
Also, I would think about taking a small fraction of the stained cells and attach them to a microscope slide using cytospin then observe them under a confocal microscope to check what is happening.
Good luck!
Yassouf
Andrew Wight I do use comp beads for controls, and I do see a clear separation between positive and negative populations, when staining with the same fluorophores I use for tissue staining.
Also - Mhd Yousuf Yassouf Andrew Wight - I forgot to mention that after collagenase digestion, I first wash cells in a hbss - phenol red buffer. When I perform the gradient separation, my 70% density layer is suspended in PBS, but the 40% layer is also hbss - phenol red.
Thinking about it, I am not sure if phenol red may be introducing additional auto fluorescence.
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