Hi,
I'm working on heart flow cytometry, and I use anti-mouse CCR2-BV785 (biolegend, cat150621) for staining, 20 minutes, 4 degree. But I couldn't see any CCR2+ cells. So I'm not sure if it's actually right or it's because of antibody not working. I just checked some literature and it's supposed to have more CCR2+ cells in heart failure.
Do you have any ideas?
Thank you so much.
Best,
Hi, I am happy to answer that for you. I did have problems with CCR stainings in the past. As antibodies bind to the extracellular loops of the CCR and the CCR is fluid and sometimes internalized it is best practice to stain CCR seperatly (after or with FC Block) at 37 degree. This improves receptor recycling to the surface and increases the chance that the loops have the right confirmation for the antibody to find the target. You can follow up with washing and staining with Mastermix. Good luck
I can recommend Laura's approach here. I stain cells first in a minimal cocktail using only CCR-specific antibodies for 10 min at 37 °C, then add all remaining antibodies of the cocktail and stain for additional 20 min in the fridge. I haven't used this particular clone though but many different human tissues.
Tobias Kammann Thank you. Did you wash cells after CCR-specific antibodies intubation? Or just add the rest of antibodies?
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