Proinflammatory cytokine release assay

PBMC release of proinflammatory cytokines was measured in vitro both in response to LPS stimulation, as well as in the absence of stimulation. After isolation, PBMCs were plated in triplicate at a density of 2.5 × 10⁵ cells/well, in a 200 µL final volume. PBMCs were plated both in media only (unstimulated condition), as well as with 1 µg/mL of LPS, obtained from E. coli (serotype 026:B6, Sigma-Aldrich, St. Louis, MO), and were incubated for up to three days at 37 °C, 5% CO₂, and 100% humidity. Cell culture supernatants were collected at 2, 24, 48, and 72 h post-plating, and then stored them at − 80 °C until assays were conducted.

Cell culture supernatants were later thawed and assayed in duplicate for levels of a trio of proinflammatory cytokines: interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) using a MILLIPLEX MAP Human Cytokine Panel magnetic bead kit (EMD Millipore Corporation, Billerica, MA), and read using a Luminex MAGPIX fluorescent detection system (Luminex, Austin, TX) and xPONENT software (Version 4.2; build: 1324; Luminex, Austin, TX). Intra-assay coefficients of variation (CVs) were 8.20% (IL-6), 6.97% (IL-1β), and 5.98% (TNF-α). Inter-assay coefficients of variation (CVs) were 17.27% (IL-6), 10.53% (IL-1β), and 11.62% (TNF-α).

Due to a freezer failure on November 5, 2016, cell culture supernatant samples were compromised for 32 participants (for additional information see⁴⁷). No compromised samples were assayed, and thus data from these samples were not included in any analysis. All other biological samples from these participants (including plasma used for the total testosterone assay) were stored elsewhere and were thus unaffected by the freezer failure.

However, given this loss of data, analyses based on these cytokine data were likely underpowered and the results should be interpreted with due caution.