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)].
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)].
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