Tried a few against hCD4, but they did not discriminate between CD4+ T cells and primary human macrophages. Both about equally as bright. Expected the T cells to be much brighter. Thought of course to use CD3, but cannot seem to find a reagent that I feel strongly will work well. Looking for suggestions from those that have used an antibody in their studies. T cells will be PHA blasts from PBMCs. Thanks for any assistance.
Just a wild guess, but can it be that the macrophages bind the antibody with the FcR?
Blocked it with Fc block and excess human IgG. Could've certainly been an issue. Thank you for the comments and assistance.
Yeah, would be too easy ;-) Another wild guess: Could you label the cells before you co-culture them? I did that once with macrophages from different genotypes with a proliferation dye ...
Why do you think CD3 co-labeling wont work? Woukd seem obvious solution. Or else label ypur macs with CD16
I agree with Florian; stain your CD4+ cells with an amin-binding proliferation stain, such as CellTrace Violet from Life Technologies, before coculture.
Could autofluorescence from the macrophages overlap with the fluorescence from the dye attached to the CD4 antibody?
How long is your co-culture? Are the macrophages M0, untreated primary macrophages or M1-M2? How healthy are the PHA blasts you are using as your CD4+ T cell source? Are you analyzing by flow or imaging? All of these parameters will impact your staining. If you enrich for your CD4+ T cells prior to co-culture with the macrophages you can label them with pHrodo and this will enable you to detect them whether or not they are associated with the macrophages (ex. engulfed). I use a pool of antibodies to stain macrophages; however, if they have been treated with (for ex.) IFN-g they will stain much better. Labeling the T cells separately and using a pool of macrophage-specific antibodies (all the same fluorophore) works well.
Macrophages are GM-CSF matured for 7 days. Plated on collagen-coated MatTek dishes. Infection performed between days 8-10 post-plating. PHA blasts are recalled from frozen since we are performing autologous experiments and HIV-1 infection of M0 takes between 8-10 days to see desired phenotype/compartment. PHA blasts could be healthier in my opinion, but cannot see a way around freezing them due to length of required macrophage infection. Thank you very much for comments. Seems you have experience in these areas and I appreciate your willingness to share it with me. Any advice you can provide is most welcome and please do not spare details.
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