Aquios cl

A fully automated flow cytometer (AQUIOS CL, Beckman Coulter, Indianapolis, IN, USA) was placed in a field laboratory. Point-of-Care flow cytometry analyses were performed on blood samples from a healthy cohort of 46 individuals [6 (link)]. A venous blood sample was collected in a sodium-heparin (Vacuette; Greiner Bio-One) blood collection tube on the day prior to and during 3 days after the day of prolonged walking exercise (20–30 km at a self-selected pace; Fig. 1). The AQUIOS CL was situated stationary in the study center next to the room where the venous blood samples were taken. Blood samples were stored at room temperature next to the machine and were analyzed with varying times between venipuncture and analysis. The time between venipuncture and analysis was accurately registered.Fig. 1

Study flowchart for the healthy cohort that was used to study ex-vivo activation of neutrophils, eosinophils and monocytes. Venous blood samples were collected from the cohort during several days around a 20–30 km walking exercise and were measured by an automated flow cytometer (see materials and methods section).

Fig. 1

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).

The identification of different cell types was performed by the flow cytometry analysis using AQUIOS CL with several antibodies (Beckman Coulter, Brea, CA, USA) by the central laboratory of the Faculty of Tropical Medicine, Mahidol University, as previously described (Gossez et al., 2017 (link)). Briefly, 50-μL of blood were stained with anti-CD45-fluorescein isocyanate (FITC), the lymphocyte common antigen, before identification as several lymphocytes using anti-CD3-proprotein convertase 5 (PC5), anti-CD4-RD1, anti-CD8-Ecdysoneless Homolog (Drosophila) (ECD), and anti-CD-19-allophycocyanin (APC). Likewise, the classical and intermediated monocytes were indicated by CD14⁺/CD16^- and CD14⁺CD16⁺, respectively, while the non-classical monocytes were CD14^-/CD16⁺. In parallel, natural killer (NK) cells were indicated by CD56⁺CD16^- cells. Then, the samples were run on a flow cytometry.