Msd discovery workbench 4

ELISAs were performed on culture supernatants using the Mouse IL-12/IL-23 p40 Non-allele-specific and Mouse IL-10 Quantikine ELISA kits (R&D Systems) according to manufacturer’s instructions. Cytokine arrays were performed on 1 ml of supernatant from the co-cultures using the Mouse Cytokine Antibody array, Panel A (R&D Systems), according to manufacturer’s instructions. IL-1β and IL-4 analysis of 24-h co-culture supernatants was performed using the MSD Mouse V-PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics, Rockville, MD). Briefly, biological triplicates of cell-free supernatants were run in duplicate and analyzed on a QuickPlex SQ120 analyzer using MSD Discovery Workbench 4.0 software (Meso Scale Diagnostics, Rockville, MD). Assay sensitivity using this platform is 0.04 pg/ml.
Whole cell lysates from differentiated muscle cultures were prepared using the NP40 Lysis buffer (Invitrogen) containing anti-proteases (Mini-Complete, Roche, Indianapolis, IN). Protein concentration was determined by the bicinchoninic acid assay (BCA) (Thermo Fisher, Waltham, MA). To assay for IL-1β, 240-ng protein per well was assayed in biological triplicates using the Mouse IL-1β Quantikine ELISA kit (R&D Systems).

Venous blood samples were collected from all participants in the morning after an overnight fast. The time of blood draw for the case group was the day after admission to the hospital. Two samples were sent to the Department of Clinical Laboratory for tests of routine blood count and blood biochemistry within 2 h. One sample was immediately sent to the Department of Clinical Laboratory for centrifugation, and separated plasma was frozen at −80°C until analysis.
Blood routine analysis was performed using an automatic hematology analyzer (Mindray BC-2800, Shenzhen, China). Plasma biochemical parameters were measured with an automatic biochemistry analyzer (AU480, Beckdman Counter, USA) by using commercial kits (Roche, Switzerland). MSD Platform (labservice.univ-bio.com, Shanghai, China) was used to measure multiplex levels of inflammation biomarkers [Flt-1, Placental growth factor (PIGF), Angiopoietin-2 (Tie-2), IFN-γ, TNF-α, TNF-β, C-reactive protein (CRP), IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-16, and IL-17A]. MSD sector 2100A was used to read the plates, and data were analyzed using MSD Discovery Workbench 4.0 software (from Meso Scale Diagnostics). The limit of detection for these inflammatory cytokines was 0.01 pg/mL, and all standards and plasma samples were assayed in a duplicate test.