Whole blood stimulation assays and cytokine quantification by ELISA

Blood sample collection, processing, and ex vivo cytokine production experiments were performed at the biotechnology laboratory facility available at Kilimanjaro clinical research institute (KCRI) in Moshi, Tanzania. A whole blood count with leukocyte differentiation was measured on a Sysmex XN-450 Hematology Analyzer (Sysmex Corporation, Kobe, Japan). Whole blood was stimulated with bacterial and fungal stimuli and TLR3 and TLR4 agonists. The stimulation experiments were performed as follows: 100 μl of heparin blood was added to a 48-wells culture plate and subsequently stimulated with 400 μl of stimulus for 48 h at 37 °C and 5% CO₂.

The bacterial and fungal stimuli were cultured and frozen at Radboudumc and then shipped to KCRI. Their respective concentrations were as follows: PHA (10 μg/ml, Sigma), LPS (100 ng/ml, Sigma), Poly:IC (50 μg/ml, Sas Invivogen), Mycobacterium tuberculosis (5 μg/ml, H37Rv, in-house), Coxiella burnetii (10⁷ CFU/ml, Nine miles/RSA493), Escherichia coli (10⁶ CFU/ml, ATCC35218, in-house), Staphylococcus aureus (10⁶ CFU/ml, ATCC29213, in-house), Candida albicans (10⁶ CFU/ml, UC820, in-house), Streptococcus pneumonia (10⁷ CFU/ml, TIGR4, in-house), Salmonella typhimurium (10⁶ CFU/ml, Phage type 510, in-house), and Salmonella enteritidis (10⁶ CFU/ml, in-house). Stimuli were prepared in RPMI culture medium (Dutch modified, Invitrogen) supplemented with 50 µg/mL gentamicin, 2 mM Glutamax, and 1 mM pyruvate.

Supernatants were collected and stored at −80 °C until used for ELISA. The concentrations of cytokines were quantified in the supernatants using ELISA according to the instructions (given IL-6, IL-1β, IL-10, and tumor necrosis factor (TNF-α): R&D Systems; interferon (IFN-γ): Sanguin). All samples were measured using kits of the same lot number.