Mouse tnf ri and tnfr 2 ultra sensitive kits

One series of brain tissue was lysed in Complete Mesoscale buffer according to the manufacturer’s protocol (Mesoscale Discovery, Rockville, MD, United States). Protein concentrations were determined according to the protocol from Micro BCA protein assay kit (Thermo Fischer Scientific). Cytokine and chemokine concentrations were estimated using an MSD Mouse Pro-Inflammatory V-Plex Plus Kit (Mesoscale Discovery) and TNF receptor concentrations using the Mouse TNF-RI and TNFR-II Ultra-Sensitive Kits (Mesoscale Discovery). XPro1595 levels were measured in ischemic brain tissue lysates using a human TNF V-Plex immunoassay (Mesoscale Discovery) as previously described (Karamita et al., 2017 (link)). The standard in the kit was replaced with XPro1595, which was diluted in the kit diluent number 2. Etanercept levels were measured using a human TNFRII Ultra-Sensitive immunoassay (Mesoscale Discovery) (Karamita et al., 2017 (link)). The standard in the kit was replaced with etanercept diluted in the kit diluent number 2. All kits were read on a SECTOR Imager 6000 plate reader (Mesoscale Discovery) according to the manufacturer’s instructions. All samples were run in duplex, and coefficient of variation (CV) values below 25% were accepted.

Plasma and brain samples were measured in the MSD Mouse Proinflammatory V-Plex Plus Kit (IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, CXCL1, TNF; K15012C, Mesoscale) and in brain samples the mouse TNF-RI and TNF-RII Ultra-Sensitive Kits (Mesoscale) using a MSD QuickPlex (SQ120) Plate Reader (Mesoscale) according to the manufacturer’s instructions5 (link). Samples were run in duplex and diluted 2- or 4-fold in Diluent 41 prior to measurement. Data was analyzed using MSD Discovery Workbench software. The lower limit of detection was a calculated concentration based on a signal 2.5 SD above the blank (zero) calibrator.