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).
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).