Duoset mouse elisas

Blood glucose was measured at the time of blood sampling using a hand-held glucometer (Freestyle Disectronic, Vianen, The Netherlands). Plasma cholesterol and triglycerides were determined using enzymatic assays (CHOD-PAP and GPO-PAP, respectively; Roche Diagnostics, Almere, The Netherlands). Plasma insulin was analyzed by ELISA (Mercodia AB, Uppsala, Sweden). Homeostasis model assessment (HOMA) was used to calculate relative insulin resistance (IR). Five hours fasting plasma insulin and fasting blood glucose values were used to calculate IR, as follows: IR = [insulin (ng/mL) × glucose (mM)]/22.5. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a spectrophotometric activity assay (Reflotron-Plus, Roche). Serum amyloid A (SAA) was analyzed by ELISA (Tridelta, Maynooth, Ireland) and both E-selectin and monocyte chemoattractant protein-1 (MCP-1) were analyzed using Duoset mouse ELISAs (R&D Systems, Minneapolis, Canada) according to the manufacturer’s instruction. Hepatic collagen content was measured via a hydroxyproline-based colorimetric assay as a marker of fibrosis using the Sensitive total collagen assay (Quickzyme, Leiden, The Netherlands).

Donor T cells were extracted from spleens of mice with aGvHD (day 7 post transplant) or control C57Bl/6 mice by negative isolation (STEMCELL Technologies) as described above. Cells were plated in media at 4 × 10⁵ cells/well in 96 well round-bottom tissue culture plates. After 4 h, supernatant was collected and used for LDH or cytokine quantification. LDH was measured using the CyQUANT^TM LDH Cytotoxicity Assay Kit (Invitrogen) and IL-1β and IL-18 levels were measured by ELISA (DuoSet Mouse ELISAs, R&D Systems). Mouse T cells isolated from C57Bl/6 mice were activated using Dynabeads Mouse T-Activator CD3/CD28 (Gibco), according to the manufacturer’s protocol, in media with IL-2. After 48 h, cells were collected and lysed for western blot analysis of caspase-1 expression.