Ifn γ

Serum levels of IDO and cytokines (TNF-α, IL-1β, IFN-γ, IL-4, IL-6, IL-17) were determined at baseline and at 6 weeks of follow-up. Venous blood was collected from the forearm between 6 and 7 a.m. following an overnight fast. Serum levels of IDO and six cytokines were measured by quantitative enzyme-linked immunosorbent assay (ELISA) using a commercially available kit: IDO (SEB547Hu, sensitivity: minimum detectable measurement ≤ 0.118 ng/mL) (Cloud-Clone, Wuhan, China), IFN-γ (SEA049Hu, sensitivity: minimum detectable measurement ≤ 6.1 pg/ml) (Cloud-Clone, Wuhan, China), IL-4 (SEA077Hu, sensitivity: minimum detectable measurement≤5.9 pg/ml) (Cloud-Clone, Wuhan, China), IL-6 (SEA079Hu, sensitivity: minimum detectable measurement ≤ 3.2 pg/ml) (Cloud-Clone, Wuhan, China), and IL-17 (SEA063Hu, sensitivity: 5.5 pg/ml) (Cloud-Clone, Wuhan, China), TNF-α (DTA00D, sensitivity: 6.23 pg/ml) (R&D Systems, USA), IL-1β (DLB50, sensitivity: 1 pg/ml) (R&D Systems, USA). All ELISA kits used in this study have been cross-reactivity validation of existing related factors to ensure their specificity. All assays were blinded to group or behavior information until after the results were finalized.

ELISA was performed on the serum of the dogs to gain more insights into the in vivo immunomodulatory properties of the ePB-MSCs in dogs suffering from OA by measuring a well-known immunomodulatory marker released by stem cells (PGE2) and pro-inflammatory cytokines (TGF-β1, C3a, IFN-γ) as well as further potential biomarkers (HA and C2C).
ELISA analysis was performed on the canine serum samples collected on day − 21, day 0, day 14 ± 1, day 28 ± 1, and day 42 ± 4 for prostaglandin E2 (PGE2), interferon-gamma (IFN-γ), transforming growth factor beta 1 (TGF-β1), hyaluronic acid (HA), complement factor C3a (C3a), and collagen type II cleavage (C2C) (Fig. 1). All ELISA experiments were performed using commercial ELISA kits (PGE2 (R&D systems), IFN-γ (Cloud clone corp.), TGF-β1 (Cloud Clone Corp.), HA (TECOmedical), C3a (Cloud Clone Corp.), and C2C (MybioSource) according to the manufacturers’ instructions. All samples were analyzed in duplicate, and the mean values were calculated and used for statistical comparison.