Mouse tgfβ1 duoset elisa kit

Serum levels of osteocalcin and TRACP5b were tested by ELISA according to the manufacturer's instructions^{12 ,15 (link),29 (link)}. Levels of TGFβ1 in mouse serum were measured using a mouse TGFβ1 DuoSet ELISA kit (#DY1679) and in human vertebral specimens using a human TGFβ1 Quantikine ELISA Kit (#DB100B) from R&D Systems according to the manufacturer's guidelines. Briefly, total TGFβ1 levels were measured in 40 μl of serum or 10 μg of vertebral protein lysates by mixing these with 20 μl 1 N HCl, followed by neutralization with 20 μl 1.2 N NaOH/0.5 M HEPES, according to the manufacturer’s sample activation procedure; levels of endogenously active TGF-β1 in these samples were measured by skipping this sample activation procedure. These samples then were diluted 1:20 in assay diluent for measurement of total TGFβ1, or they were diluted 1:4 in assay diluent for measurement of active TGFβ1. ELISA plates were analyzed by investigators blinded to sample identity by reading absorbance using a microplate reader set to 450 nm with wavelength correction set to 540 nm.

Mouse cytokine Th1/Th2 kit from BioRAD (Bio-Plex Pro™ Mouse Cytokine Th1/Th2 Assay #M60-00003J7) and mouse TGF-β1 DuoSet ELISA kit from R&D systems (DY-1679-05) was used to determine the secretory cytokines from Gr-1⁺CD11b⁺ transduced with miR-130a and miR-145 lentivirus vectors or miRNA mimics. These transduced cells were cultured in serum-free medium for 24 h, and supernatants from the cells were collected. The amount of secreted cytokines and the ratio of M1/M2 was calculated by dividing each M1 cytokine (TNFα, IL-12, GM-CSF) to M2 cytokine (TGFβ1, IL-10, IL-4)^{37 (link)}. Briefly, each individual cytokine was measured by Bio-plex, and each individual M1/M2 ratio (TNFα/TGFβ1, TNFα/IL-10, TNFα/IL-4, IL-12/TGFβ1, IL-12/IL-10, IL-12/IL-4, GM-CSF/TGFβ1, GM-CSF/IL-10, and GM-CSF/IL-4) was calculated, and summed up for each of miRNA and control. For preventing the mRNA degradation of TβRII, IGF1R and IRS1 in Gr-1⁺CD11b⁺ cells by miR-130a or miR-145, overexpression constructs without 3′-UTR were utilized including FerH-mouse Tgfbr2 (Protein Expression Laboratory in Frederick National Laboratory for Cancer Research), pReceiver-mouse Igf1r (GeneCopoeia), pBABE-mouse Irs1 (Addgene). The M1 and M2 cytokine ratio was calculated as indicated above.

IL-10, IL-17a, IL-21 and TGFβ1 were measured using the ELISA MAX™ kit (BioLegend), Ready-SET-Go! ELISA kits (eBioscience) and mouse TGFβ-1 DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), respectively, according to the protocols provided by the manufacturers and the results were analyzed on a microplate spectrophotometer (Perkin Elmer).