Mouse erythropoietin quantikine elisa kit

Cell culture supernatant were stored in a − 80 °C freezer after collection, transferred into a − 20 °C freezer 12–16 h prior to analysis and thawed on ice before analysis. The analysis was performed according to the protocol of provided in the Mouse Erythropoietin Quantikine ELISA Kit (R&D Systems).

Mice were injected intraperitoneally with 80 ng of purified α-toxin. Peripheral blood was obtained 24 hours after the injection via the vena cava using heparinized syringe. A mouse erythropoietin Quantikine ELISA kit (R&D Systems) was used to measure plasma EPO levels. The procedures were performed in accordance with the manufacturer’s instructions.

The plasma EPO level was measured using the Mouse Erythropoietin Quantikine ELISA kit (#MEP00B; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. In brief, thawed plasma was diluted 2-fold by calibrator diluents and then incubated on an antibody-coated microplate with each volume of assay diluents for 2 h using an orbital shaker. Washed wells were treated with antiserum conjugate for 2 h and with a substrate mixture for 30 min. The optical density measured at 450 and 540 nm was analyzed with the standards curve using the 4-parameter logistic curve-fit.

The bioactivity of EPO secreted from the EPO-overexpressing NIH/3T3 cell line was examined in a cell model of neurodegeneration, PC12-INT-EGFP cells. 3T3, 3T3-EGFP, and EPO-3T3-EGFP cells (1.6 × 10⁶ cells) were seeded in 10 cm dishes and cultured for 24 h. Culture supernatants were collected by sterile syringes and filtered through hydrophilic polyethersulfone membranes with a pore size of 0.22 μm (Millipore) for subsequent functional assays. The concentration of secreted EPO in collected culture supernatants was also measured using the Mouse Erythropoietin Quantikine ELISA Kit (R&D Systems). After 6 days of NGF induction, PC12-INT-EGFP cells were supplemented with conditioned media (50% v/v), which combined half of the fresh medium for PC12 cells and half of the collected culture supernatants, as mentioned previously. Moreover, 10 IU/mL of recombinant human EPO (hrEPO; specific activity, 115,336 IU/mg P; Millipore) was applied as the positive control. PC12 cells treated with fresh medium alone were used as the vehicle group. A cell viability assay, 48 h live-cell imaging, and immunocytochemistry were performed on PC12-INT-EGFP cells for testing the bioactivity of the secreted EPO.