Mouse tgf β1 quantikine elisa kit

The primary cells were compressed for 4 h followed by 12 h of uncompressed incubation. The conditioned medium was collected to determine the concentration of total and active TGF‐β1 using Mouse TGF‐β1 Quantikine ELISA Kit (MB100B, R&D Systems). According to the manufacturer's instructions, active TGF‐β1 was measured directly from the conditioned medium, whereas total TGF‐β1 was measured from the conditioned medium with acid treatment.
Western blot analyses were conducted on the protein of lysates from the primary cells compressed for 4 h. The supernatants of lysates were collected after centrifugation and separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and then blotted on the nitrocellulose blotting membranes (MilliporeSigma, Burlington, MA). The primary antibody for Thbs1 (1:1000, ab267388, Abcam) and α‐Actinin (1:1000, 3134, Cell Signaling Technology, Danvers, MA) was applied for incubation, and the proteins were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

Serum collection was performed on day 0, prior to tumor inoculation, and at indicated time points (i.e., day 14 and end of study). Serum IL-1β, IL-2, IL-5 and TNFα were quantified using the murine V-Plex Proinflammatory Panel 1 kit and MESO QuickPlex SQ 120 (Meso Scale Diagnostics; Rockville, Maryland, USA), according to the manufacturer’s instructions. TGFβ levels were quantified using mouse TGFβ1 Quantikine ELISA kit according to the manufacturer’s instructions (R&D Systems; Minneapolis, Minnesota, USA). Data presented herein represent the change in cytokine, with each animal’s baseline levels subtracted from subsequent timepoints.

The levels of TGF-β1, collagen I, and collagen III in the liver and serum samples were determined using commercially available ELISA kits including Mouse TGF-β1 Quantikine ELISA kit, Mouse Collagen type I (Col I) ELISA kit, and Mouse Collagen type III (Col III) ELISA kit (R&D Systems, Minneapolis, MN). The serum level of TGF-β1 was also determined using a commercially available ELISA kit (R&D Systems, Minneapolis, MN).