Serum separator clot activator blood collection tubes

Blood samples (4 mL) from participants were collected in serum separator clot activator blood collection tubes (Greiner Bio‐One; reference no. 454071). The blood samples were allowed to rest upright on the laboratory bench for 30 minutes before centrifugation at 5000 g for 10 minutes at room temperature. Approximately 2 mL of supernatant sera was harvested by pipette, frozen, and stored at −80°C in polypropylene cryogenic vials. Following a complete thaw, resting levels of proinflammatory cytokines were measured using a mesoscale discovery (MSD) platform (Meso Scale Discovery). An ultrasensitive human proinflammatory I, V‐Plex immunoassay panel was used to measure serum levels of interleukin‐6 (IL‐6), IL‐8, IL‐10, and tumor necrosis factor alpha (TNF‐α). Samples were diluted 1:2 according to the manufacturer's protocol. The lower limit of detection was < 1 pg/mL for all assays, and standardized calibration curves were confirmed before testing. All serum samples were measured in duplicate, and the mean cytokine concentration of the duplicates (in picograms per milliliter) was used for analysis.

Blood samples (4 ml) from participants were collected in serum separator clot activator blood collection tubes (Greiner Bio-One, Stonehouse, United Kingdom; reference no. 454071). The blood samples were allowed to rest upright on the laboratory bench for 30 min before centrifugation was performed at 1,000 × g for 20 min at room temperature. Approximately 2 ml of supernatant sera was then harvested by pipette, frozen, and stored at −80°C in polypropylene cryogenic vials. At a later date, following a complete thaw, resting levels of proinflammatory cytokines were measured using a mesoscale discovery (MSD) platform (Meso Scale Discovery, Rockville, MD). The MSD system is an electrochemiluminescence-based solid-phase multiplex assay. An ultrasensitive human proinflammatory I, V-Plex immunoassay panel containing interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IL-10, and gamma interferon was used to measure serum levels. Samples were diluted 1:2 according to the manufacturer’s protocol, and samples from all 3 intervention groups were dispersed across each MSD plate. The lower limit of detection was <1 pg/ml for all assays. All plasma samples were measured in duplicate, and the mean cytokine concentration of the duplicates (in picograms per milliliter) was used for analysis.