V plex proinflammatory panel 1 mouse

The levels of PELF cytokines (TNFα, IL-6, and IL-10) were evaluated by using mouse cytometric bead array (CBA) cytokine (BD Biosciences) and V-PLEX Proinflammatory Panel 1 Mouse (Meso Scale Discovery) multiplex kits according with the manufacturer's instructions. Readings were compared with those of appropriate standard curves, and results are expressed as picograms of cytokine per millilitre of PELF (pg/ml).

Cytokine and chemokine concentration was quantified in BALF and serum samples using a V-PLEX® Cytokine Panel 1 (mouse) and V-PLEX® Pro-inflammatory Panel 1 (mouse) (Meso Scale Diagnostics, LLC, Rockville, MD). These panels included detection of MCP-1 (CCL2), MIP-2 (CXCL2), IL-1β, IL-6, KC/GRO, IL-10, and TNFα. Detection was performed according to the manufacturer’s specifications on a MSD SQ120MM (VB204) instrument, with curves analyzed by Discovery Workbench® using a 4PL Curve fit for all analytes. Samples were assayed in duplicate by Crux Biolabs (Bayswater, VIC, Australia).

Spleens were excised and single cell suspensions were obtained and re-stimulated as previously described (85 (link)). Cells were washed and re-suspended in complete medium [RPMI 1640 supplemented with 10% FCS, 1% glutamine, 1% penicillin-streptomycin, 1% Na-pyruvate, 1% non-essential amino acids (Gibco, Life Technologies GmbH) and 50µM 2-β-mercaptoethanol (Sigma-Aldrich)], plated in 96-well at a concentration of 2x10⁵ cells/well and cultured for 72 h with medium alone or with OVA V (5 µg/ml; Sigma-Aldrich). Their supernatants were analyzed for cytokine expression using the V-Plex proinflammatory panel 1 mouse and V-plex IL-9 kit (MesoScaleDiscovery) according to manufacturer’s instructions.