Cd163 bv421

Lidocain (4 ng/mL) was added to macrophages and then cells were detached by gentle scraping, spun down, and collected in buffer (PBS plus 0.5% bovine serum albumin). Cells were incubated for 20 min at 4°C with appropriate antibodies, fluorescence minus one (FMO), and single stains were taken along for every antibody. Antibodies were as follows: CD14-AF700, CD68-PE-CY7 CD86-BV650, CD163-BV421, CD209-FITC, CD206-APC (Biolegend, San Diego, CA, USA). The cells were then washed twice with 100 μl of flow buffer, centrifuged, and resuspended in 200 μl flow buffer for analysis. Flow cytometry was performed with fluorescence-activated cell sorter (FACS) Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). The cell populations were gated based upon FSC and SSC parameters and normalized to cells alone (without antibody) to adjust for cell-specific autofluorescence and a gate was set on the CD14 positive population (Supplementary Figure 4). Data were analyzed using FlowJo (FlowJo JCC, Ashland, OR, USA).

The following anti-human antibodies were used for staining: CD68-FITC, CD14-PercP-Cy5.5, HLA-DR-APC, CD15-PE-Cy7, CD11b-PErcP-Cy5.5, CD163-BV421, CD14-APC, HLA-DR-PE, CD33-BV421 and PD-L1-BV421 purchased from Biolegend (San Diego, CA), CD11b-PE, CD3-Alexa Fluor 700, CD19-Alexa Fluor 700, CD19-Alexa Fluor 700, CD163-Alexa Fluor 647, CD16-PE-Cy7, CD3-PE, CD19-PE and CD56-PE purchased from BD Biosciences (San Jose, CA), CD14-PE-TR and CD16 PE-TR purchased from Life Technologies (Carlsbad, CA) and CD86-FITC purchased from R&D systems (Minneapolis, MN). CD32-a-FITC Ab was purchased from Stemcell technologies, (Vancouver, Canada). CD-32-B (F-4) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, and secondary Ab anti-mouse F(ab’)2 was purchased from Thermo fisher scientific (MA, USA).
Intracellular staining of CD68 was performed as follows: PBMC were stained with surface marker antibodies, fixed with fixation/permeabilization buffer (eBioscience), washed, and stained for intracellular antigens in 1X permeabilization buffer. Cells were analyzed on an LSR Fortessa (BD) flow cytometer, and data analyzed using Flow Jo (Treestar, Ashland, OR). Dead cells were excluded based on viability dye staining (Zombie Aqua Fixable Viability Dye, Biolegend).

Apoptosis was detected using the Annexin V and Propidium Iodide (PI)/7-AAD Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells were stained with PI (Sigma, St Louis, MO, USA) for cell cycle analysis and CD14-FITC, CD11b-APC, CD86-PE, and CD163-BV421 (BioLegend, San Diego, CA, USA) for differentiation analysis. All the preparation work before flow cytometric analysis was performed according to the corresponding manufacturer’s instructions. Flow cytometric data were collected using an LSR II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software V10 (Becton Dickinson, Franklin Lakes, NJ, USA).