Anti ova

To generate the six macrophage phenotypes, we sampled the cultured RAW 264.7 cells into 6 well plates with concentration of 0.25 × 10⁶ cells/ml having a total volume of 2 mL of the culturing media. The unstimulated RAW 264.7 were considered as M0 (naïve) macrophages, while M1, M2a, M2b, M2c, M2d polarization states were respectively achieved by applying the following treatments: (i) 25 ng/mL IFN‐γ (Genesearch, #39127S) for 24 h and 25 ng/mL LPS for 24 h were used to generate M1 phenotype; (ii) 25 ng/mL IL-4 (Genesearch, #5208SC) for 24 h to generate M2a phenotype; (iii) 200 µL of immune complexes and 50 ng/mL LPS for 24 h were added to generate M2b phenotype. Immune complexes were made as previously described^{67 (link)}, briefly, 14 µg of OVA (Sigma-Aldrich, #A5503) was incubated with 39 µL anti-OVA (Sigma-Aldrich, C6534) for 30 min, using rotation at room temperature; (iv) 50 ng/mL IL-10 (Genesearch, #5261SC) and 50 ng/mL of TGF-β (Genesearch, #5231LF ) for 24 h to generate M2c phenotype; (v) LPS 50 ng/mL and 50 μM 5′-(N-thylcarboxamido) adenosine (NECA) (Sigma-Aldrich, #E2387) for 24 h were used to generate M2d phenotype. All polarization experiments were conducted on adherent macrophages, as culturing on the low adherence plates affects macrophage polarization^{68 (link)}.