Dy240e

The PF-4, TGF-β₁, and, PDGF-BB concentrations from each blood component cultured with each bacterium were determined in duplicate by sandwich ELISAs developed with commercial antibodies for human PF-4 (human CXCL4/PF-4, DY795; R&D Systems, Inc., Minneapolis, MN, USA), TGF-β₁ (human TGF-β₁, DY240E; R&D Systems, Inc.), [28 (link)] and PDGF-BB (human PDGF-BB, DY220; R&D Systems, Inc.) [29 (link)]. The thresholds of detection were 15.6 pg/mL for PF-4, 31.2 pg/mL for TGF-β₁, and 31.2 pg/mL for PDGF-BB. The ELISAs were performed according to the manufacturer's instructions. Readings were made at 450 nm [13 (link)].
A 2.5 mL sample of PRP and LPP from each horse was incubated at 37°C for 15 minutes with 250 μL of a solution containing 0.5% of a nonionic detergent (NID) (TritonX100, Sigma-Aldrich Co. LLC., MO, USA). Both blood components treated with NID were used as a positive control of PF-4 and GF release [30 (link), 31 (link)]. It is important to consider that TGF-β₁ and PDGF-BB have been measured in equine PRP by using human ELISA antibodies [13 (link), 27 (link), 32 (link)].

The TGF-β₁ and PDGF-BB concentrations from the supernatants and lysates of each blood component were determined in duplicate by a sandwich ELISA using commercially available antibodies against human TGF-β₁ (Human TGF-β1, DY240E, R&D Systems, Inc., Minneapolis, MN USA) and PDGF-BB (Human PDGF-BB, DY220, R&D Systems, Inc.). Both ELISAs were performed according to the manufacturer’s instructions. Readings were performed at 450 nm. Both ELISAs were determined with human antibodies because there is a high homology of these growth factors between equines and humans [18 (link),19 (link)]. Further, several equine PRP studies have validated these ELISA kits [6 (link),14 (link)-16 (link)].