Duoset elisa ancillary reagent kit 2

Frozen tissue homogenates (from 12-25 weeks old mice) described above were thawed, total protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Scientific #23227) and the samples were adjusted to equal protein concentration. ELISA was performed according to manufacturer’s instructions using IL-1 beta Mouse Uncoated ELISA Kit, MIP-2/CXCL2 Mouse ELISA Kit, MIP-1a (CCL3) Mouse Uncoated ELISA Kit (Invitrogen, ThermoFisher Scientific, catalog numbers 88-7013-88, EMCXCL2, and 88-56013-88), Mouse IL-17A/F Heterodimer DuoSet ELISA, and Mouse CXCL1/KC DuoSet ELISA DY5390-05, and DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems, catalog numbers DY5390-05, DY453-05, DY008).

Blood samples drawn from the antecubital vein of all participants for the measurement of plasma DAMPs (S100B, S100A12, HMGB1, HSP70, DJ-1 and their receptors RAGE and TLR4) occurred in the morning before the coronary angiography to avoid interference with the inflammatory status due to the plausible interventions that might occur during the catheterization process.
All samples were centrifuged at 3200× g for 10 min at a temperature of 4 °C, within an hour after collection. The plasma was immediately separated into aliquots to avoid loss of bioactive cytokines, and rapidly stored in −80 °C, until the assay analysis. The plasma concentrations of human sRAGE, DJ-1, S100B, S100A12, HSP70, TLR4, and HMGB1 were quantified using the respective DuoSet ELISA kit together with the DuoSet ELISA Ancillary Reagent Kit 2 according to the manufacturer’s instructions (R&D Systems Inc., Minneapolis, MN, USA). The sensitivity for each kit was as follows: sRAGE (sensitivity—16.14 pg/mL), DJ-1/Park7 (sensitivity—62.5 pg/mL), S100B (sensitivity—50 pg/mL), S100A12 (sensitivity—7.8 pg/mL), and HSP70 (sensitivity—125.0 pg/mL), TLR4 (sensitivity—31.25 pg/mL), and HMGB1 (sensitivity—18.75 pg/mL) (Novus Biologicals, Centennial, CO, USA).

The levels of TNF, IL-1β, IL-6, IL-10, keratinocyte chemoattractant (KC/CXCL1), monocyte chemoattractant protein-1 (MCP-1/CCL2), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), and eotaxin-1/CCL11 were measured by Mouse DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA), and the level of macrophage inflammatory protein-2 (MIP-2/CXCL2) was determined using the Mouse MIP2 Matched Antibody Pair Kit (Abcam, Cambridge, UK). The ELISA kits from both manufacturers were used in combination with the DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems). All assays were performed according to the manufacturers’ instructions with only minor modifications, and optical densities at 450 and 540 nm were measured on the Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA).