Tgf β elisa kit

2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrobenzenesulphonic acid (DNSB), Phorbol 12-myristate 13-acetate (PMA), concavalin A (Con A), indomethacin, ionomycin and monensin were purchased from Sigma Aldrich (Brøndby, Denmark). rmIL-1β, rmIL-23, IFN-γ, IL-10, TGFβ ELISA kits, fixable viability dye eFluor780, and intracellular staining kit was purchased from eBioscience (San Diego, CA, USA). Mouse TH1/TH2 9-plex and mouse IL-17A Ultra-sensitive kits were purchased from Meso Scale Discovery. Anti-CD16/CD32 (2.4G2), anti-γδ (GL3), anti-Vγ3 (536), anti-CD3ε (145–2C11), anti-IL17 (TC11-18H10), anti-IFN-γ (XMG1.2) and isotype control (R3–34) were all purchased from BD Pharmingen. Anti-CD25 (PC61) was purchased from Biolegend, San Diego, CA, USA. TaqMan Reverted Aid First Strand cDNA synthesis Kit (K1622), TaqMan Universal PCR master mix (4326708) and primers GAPDH (Mm999999_g1) and IL-1β (Mm01336189) were purchased from Applied Biosystems.

Active TGFβ concentrations in media and cell cultures treated with Vehicle, CR peptide, and CR control were measured using a TGFβ ELISA kit (88–6350, Thermo Scientific). Standards and sample reagents were prepared per the manufacturer’s instructions. TGFβ activity in Col3^−/− fibroblasts was measured from cells plated on silicone overnight followed by incubation in complete media with the CR peptide or CR control for 48 hrs, as described for silicone wrinkling. Cells were collected in lysis buffer (RIPA, Cell Signaling) with protease and phosphatase inhibitors (P8340 and P0044; Sigma-Aldrich) for use in the ELISA. Cell lysates were the same used for immunoblotting. Peptide interactions with TGFβ were done by incubating 500 nM of CR peptide or CR control together with human TGFβ1 (2ng/mL; 240B; R&D Systems) for 30 min at 37°C. Samples were stored at −80°C. Measurements were made using a plate reader (Pierce, GloMax) at a wavelength of 450 nm, and compared to the TGFβ standard curve for quantification.