Sequential Staining Improves Detection of CCR2 and CX3CR1 on Monocytes When Simultaneously Evaluating CCR5 by Multicolor Flow Cytometry

Using multicolor flow cytometry, we sought to evaluate and optimize the simultaneous detection of cell surface expression levels of CCR2, CX3CR1 and CCR5 in primary human monocytes using a single antibody panel.
Conclusion:
We observed that staining for CCR2 alone or for CX3CR1 alone showed greater expression levels than when the cells were stained with the full panel of antibodies. Fluorescent-minus-one (FMO) controls revealed that ligation of the CCR5 monoclonal antibody to the receptor interfered with detection of CX3CR1 and CCR2. Sequential addition of the antibodies during the staining procedure was sufficient to restore the detection levels, suggesting close proximity and possible functional interactions between CCR2/CCR5 and CX3CR1/CCR5 in monocytes. Fluorescence-Minus-One (FMO) and single-stained samples.