HO-1 (human), ELISA kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
All reagents are stable as supplied at 4°C, except the Recombinant HO-1 Standard, which should be stored at -20°C. For optimum storage, the Recombinant HO-1 Standard should be aliquotted into smaller portions and stored at -20°C.	-Include a standard curve each time the assay is performed. -Mix all reagents and samples gently, yet thoroughly, prior to use. Avoid foaming of reagents. -In this protocol, room temperature refers to 20-28C. The room temperature should remain within this range throughout the assay. -Consistent, thorough washing of each well is critical.

Upstream tips
All reagents are stable as supplied at 4°C, except the Recombinant HO-1 Standard, which should be stored at -20°C. For optimum storage, the Recombinant HO-1 Standard should be aliquotted into smaller portions and stored at -20°C.
Protocol tips
-Include a standard curve each time the assay is performed. -Mix all reagents and samples gently, yet thoroughly, prior to use. Avoid foaming of reagents. -In this protocol, room temperature refers to 20-28C. The room temperature should remain within this range throughout the assay. -Consistent, thorough washing of each well is critical.

Publication protocol

Enzyme-linked immunosorbent assays (ELISA) were conducted according to the manufacturers’ instructions to measure plasma concentrations of haptoglobin (HPT, GWB-8DA44B, Genway Biotech), haemopexin (HPX, GWB-4B6D1A, Genway Biotech), haem oxygenase-1 (HO-1, ADI-EKS-800, Enzo Life Sciences), transferrin (Tf, ab108911, abcam), and ferritin (ab108837, abcam). Colorimetric determination of haem in plasma samples was conducted according to the manufacturer’s instructions (DIHM-250, Bioassay Systems). Samples were diluted prior to testing as follows: 1:50,000 (HPT); 1:1 (haem), 1:40,000 (HPX), 1:10 (Ferritin), 1:6.6 (HO-1), and 1:200 (Tf). Plasma cytokine concentrations were determined by magnetic bead multiplex assay (EPX01A-10228-901 (IFN-γ), EPX01A-10215-901 (IL-10), EPX01A-12001-901 (G-CSF), EPX01A-10213-901 (IL-6), EPX01A-10223-901 (TNFα), EPX01A-10224-901 (IL-1β), EPX01A-10225-901 (IL-4), EPX01A-10204-901 (IL-8), ProcartaPlex Multiplex Immunoassay, Invitrogen, USA) following the manufacturer’s instructions, and analysed on a Luminex 100 (LuminexCorp, Austin, USA) running Bioplex Manager software. Samples were diluted 1:2.5. Samples giving values below the limit of detection were arbitrarily assigned a concentration at the limit of detection for the purposes of statistical analyses (13.008 pg/mL for IFN-γ, 9.5146 pg/mL for IL-10).

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Manufacturer protocol

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