Dy220

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)).

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)].