Alexa fluor 488 anti human cd16

CBC and T-cell analyses were performed by Karolinska University Laboratory, Stockholm, Sweden, using Sysmex XN-9000 for CBC processing. T-cell analysis for CD4+ and CD8+ expression was performed using an Aquios CL (Beckman coulter) which utilizes a direct volumetric single‐platform method with incorporated sample preparation with a monoclonal antibody mixture (anti‐CD45‐FITC [clone B3821F4A], anti‐CD4‐RDI [clone SFCI12T4D11], anti CD8‐ECD [SFCI21thyD3], anti‐CD3‐PC5 [clone UCHT1]) Beckman Coulter.
For PBMC isolation, whole blood was sampled using BD Vacutainer^® CPT™ Mononuclear Cell Preparation Tubes and processed within 3 hours of collection. PBMCs were then isolated according to the standard procedure (centrifuged at 1500 g for 20 min at room temperature) and washed with cold PBS (440 g for 10 min at 4°C). Single cell suspensions were plated in 96-well V-bottomed plates and stained for 20 min at 4°C. The cells were incubated with Alexa Fluor647 anti-human CX3CR1 (clone: 2A9-1, BioLegend), PerCP/Cy5.5 anti-human CD192 (CCR2) (clone: K036C2, BioLegend), APC/Cy7 anti-human CD68 (clone: Y1/82A, BioLegend), PE/Cy7 anti-human CD11b (clone: ICRF44, BioLegend), Alexa Fluor488 anti-human CD16 (clone: 3G8, BioLegend) and PE anti-human CD14 (clone: 63D3, BioLegend). Cells were acquired using a Gallios flow cytometer (Beckman Coulter) and analyzed using Kaluza software (Beckman Coulter).

Monocytes were washed in 1× PBS and incubated in blocking solution consisting of FACS buffer (145 mM NaCl, 8.45 mM Na₂HPO₄, 1.83 mM NaH₂PO₄, and 0.1% NaN₃), 5% BSA, and human FcR binding inhibitor (eBioscience, San Diego, CA, USA) for 20 min on ice. After blocking, cells were stained by adding APC anti-human CD14, Alexa Fluor 488 anti-human CD16, PE/Cy7 anti-human CD163, Brilliant Violet 605 anti-human CD86 (BioLegend, San Diego, CA, USA), and APC-R700 anti-human CD80 antibodies (BD Biosciences, San Jose, CA, USA) or isotype controls on ice. Cells were then washed in 1× PBS and analyzed by flow cytometry using an LSRFortessa cell analyzer and BD FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA).
For viability studies, monocytes were washed after incubation with the APC anti-human CD14 antibody as described above. Cells were then stained with phycoerythrin-annexin V (BD Pharmingen, Franklin Lakes, NJ, USA) and either Sytox Blue dead cell stain (Life Technologies, Carlsbad, CA, USA) or propidium iodide (PI) dead cell stain (Thermo Fisher Scientific, Waltham, MA, USA) to detect dead and dying cells. After staining, cells were analyzed by flow cytometry as described above. Double negative cells represent live cells, whereas double positive and single positive cells represent dead and/or dying cells.