Vacutainer cpt mononuclear cell preparation tube

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).

At each of the study sites, venous blood was collected from consented participants into BD Vacutainer CPT Mononuclear Cell Preparation Tubes (BD Biosciences) with sodium citrate. Tubes with separated blood samples were centrifuged for 10 minutes at 2000 rpm on site, and then stored on ice for ~ 8 hours during transportation to the University of Florida research lab in Gressier, Haiti. Both plasma and PBMCs were then separated via further centrifugation for 10 minutes at 2000 rpm. Plasma was decanted and stored at -80°C. Cells were enumerated using 0.4% trypan blue (Thermo Fisher Scientific) and a hemacytometer (Hausser Scientific) and stored at -80°C in RPMI 1640 with L-glutamine and 25mM HEPES (Corning Mediatech) supplemented with 10% heat-inactivated Hi-FBS (Gibco Life Technologies) and Penicillin/Streptomycin solution (Gibco Life Technologies) with 10% molecular grade DMSO. Samples were shipped on dry ice via air courier to the Emerging Pathogens Institute at the University of Florida, Gainesville, FL. Upon arrival to the Emerging Pathogens Institute, plasma was stored at -80°C and PBMCs were stored in liquid nitrogen until used in experiments.

Blood from patients and healthy controls were collected using BD Vacutainer^© CPT™ Mononuclear Cell Preparation Tubes (BD Biosciences; Cat.362761) and spun at 1650 RCF for 15 min to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood samples from patients and healthy control after density centrifugation. Cells were then washed twice with 1× PBS (Gibco^©, Cat. 10010023) at 500 g for 5 min. PBMCs were resuspended in 10 million cells per mL in freezing media (FBS + 10% DMSO; Sigma‐Aldrich, Cat. D2650‐5X5ML) and cryopreserved. Cryopreserved PBMCs were thawed at 37°C, and washed twice with 1x PBS at 500 g for 5 min. As per the manufacturer's protocol specifications, CD4⁺ T cells were then isolated from PBMCs using an EasySep™ Human CD4⁺ T cells enrichment kit (STEMCELL™, Cat. 19052). Isolated CD4⁺ T cells were washed twice with 1× carrier‐free PBS (Rockland, Cat. MB‐008) and resuspended in 1–2 million cells per mL for staining.