HuH-7 and Hep G2 cells (3 × 105 cells) were seeded on six-well plates. The cells were incubated with treatment for distinct doses and time periods, as indicated in Figures 3b and c . For immobilized ConA treatment, beaded-agarose ConA (G-Biosciences, St Louis, MO, USA) were treated as indicated in Figures 6b and c . The supernatants were collected and the levels of secreted MIF were detected by human MIF sandwich enzyme-linked immunosorbent assay (MIF-ELISA) according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). The absorbance value at 450 nm was read by a VersaMax microplate reader.
Mif elisa
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The MIF ELISA is a quantitative sandwich enzyme-linked immunosorbent assay designed to measure the concentration of MIF (Macrophage Migration Inhibitory Factor) in biological samples, such as cell culture supernatants, serum, and plasma.
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3 protocols using mif elisa
1
Quantifying Secreted MIF in Cell Lines
2
Characterizing Hematopoietic Cell Phenotypes
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Anti-HIF1α antibody was purchased from BD Biosciences (Oxford, UK). Anti-HIF2α was purchased from (Novus Bio, Cambridge, UK Cat: NB100-122SS) IgG-FITC, IgG-PE, IgG-APC, anti-CD34-PE, anti-CD33-APC and anti-CD45-FITC antibodies were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany Cat. 130098847, 130098845, 130092214, 130098139, 130098043, 130098864). Recombinant human MIF, and MIF ELISA were purchased from R&D Systems (Abingdon, UK). All other reagents were obtained from Sigma-Aldrich (St Louis, MO, USA), unless otherwise indicated
3
Hypoxia Signaling Pathway Analysis
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Briefly, whole cell lysates were extracted and SDS PAGE gel electrophoresis separation performed as previously described (42) . Western blot analysis performed with anti-HIF1α and anti-HIF2α antibody, membranes were re-probed for B-actin as a loading control. MIF chemokine expression in the media we used target-specific MIF ELISA (R&D Systems).
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