PBMCs were extracted using the standard Ficoll gradient centrifugation method (Ficoll® Paque Plus, GE Healthcare). Cell suspensions were centrifuged, and cell pellets were blocked with fluorescence activated cell sorting (FACS) buffer with 2% mouse serum (Sigma) per 0.5–1 × 106 cells. Following blocking for 30 min, the PBMCs were stained with a panel of relevant conjugated antibodies including [CX3CR1-APC and CCR2-PE (Biolegend)] or appropriate isotype controls [Rat IgG2b κ-APC and Mouse IgG2a κ-PE (Biolegend)] and incubated at 4°C for 30 min. Following incubation, the PBMCs were washed and then fixed with 2% paraformaldehyde (PFA) and re-suspended in FACS buffer for flow cytometry. Flow cytometry was performed using the BD LSR Fortessa machine with BD FACS Diva software.
Monocytes were gated as described in the literature (22 (link)) (Supplementary Figure4 ) and a minimum number of 10,000 monocyte events were collected per sample. PBMCs from healthy controls, labeled with single conjugated antibodies, were used to determine the appropriate compensation for spectral overlap of fluorophores.
Flow cytometry data was analyzed using Flow Jo software, version 10. The percentage of positive cells and marker expression levels were determined with reference to isotype control samples (Median Fluorescence Intensity (MFI) Test/Isotype ratio).
Monocytes were gated as described in the literature (22 (link)) (Supplementary Figure
Flow cytometry data was analyzed using Flow Jo software, version 10. The percentage of positive cells and marker expression levels were determined with reference to isotype control samples (Median Fluorescence Intensity (MFI) Test/Isotype ratio).