Human tgf beta 1 duoset elisa

TGF-β1 and PDGF-BB were measured after activation in both PPP and PRP fractions to assess the effect of the centrifugation in PLTs and associated GFs. TGF-β1 and PDGF-BB were measured using ELISA kits (Human TGF-beta 1 DuoSet ELISA de R&D Systems DY240-05 and Human PDGF-BB DuoSet ELISA de R&D Systems DY220, respectively) according to manufacturer's instructions. These GFs were determined using human antibodies because these kits have been used for the same purposes in other feline PC studies (20 (link), 44 (link)) since it has been reported that human and cat PDGF-BB present high peptide sequence homology (45 (link)).

Medium was collected from hydrogel cell constructs on days 3, 6, 9, and 12 of culture and stored at − 80 °C until analysis. Total TGF-β1 levels were assessed using Human TGF-beta 1 DuoSet ELISA (DY240; R&D Systems, Minneapolis, MN). Similarly, GDF-15 expression was analyzed using Human GDF-15 DuoSet ELISA (DY957; R&D Systems). For the activation of latent TGF-β1, the medium was treated with Sample Activation Kit (DY010; R&D Systems). TGF-β1 and GDF-15 ELISA were performed using Substrate Reagent Pack (DY999; R&D Systems), and absorbance measurements were made at 450 nm with a 570 nm correction using a SpectraMax i3x Multi-Mode Microplate Reader. α-Amylase expression was quantified using the Human Salivary Amylase Alpha ELISA Kit (NBP2-68203; Novus Biologicals, Littleton, CO), and luminesce measurements were made using a SpectraMax i3x Multi-Mode Microplate Reader. All assays were performed in accordance with the manufacturers’ protocols. Protein levels were normalized to the number of cells in each hydrogel at each time point using the procedure detailed in the 3D Proliferation method conducted on days 1, 3, 6, 9, and 12 of culture.

Serum levels of Th1 (TNF-α and IFN-γ), Th2 (IL-4), Th17 (IL-6 and IL-17A), and Treg (IL-10) cytokines were assessed by flow cytometry (BD FACSCanto II cytometer, BD Cytometric Bead Array Human Th1/Th2/Th17 Kit, USA). The kit included beads coated with capture antibodies and PE-conjugated antibodies specific for IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A. The detection limits of the assay were 2.6 pg/mL for IL-2, 4.9 pg/mL for IL-4, 2.4 pg/mL for IL-6, 4.5 pg/mL for IL-10, 3.8 pg/mL for TNF-α, 3.7 pg/mL for IFN-γ, and 18.9 pg/mL for IL-17A. Data processing was performed using the FCAP Array 3.0 software (BD, USA). Serum concentrations of Th17 cytokine IL-21 (Human IL-21 DuoSet ELISA, R & D Systems, USA) and Treg cytokine TGF-β1 (Human TGF-beta 1 DuoSet ELISA, R & D Systems, USA) were determined using PowerWave spectrophotometer (BioTec, USA). The detection limits of the assay were 15.6 pg/mL for IL-21 and 31.2 pg/mL for TGF-β1. The predominant serum Th cytokine profile was determined by calculating the ratios between serum levels of cytokines associated with Th1 and Th17 cell subpopulations.