PBMCs were isolated from three thoroughbred horses: an 8-year-old gelding, a 12-year-old gelding, and an 18-year-old mare. The Institutional Animal Care and Use Committee (IACUC) of Cornell University approved the use of horses in these studies (IACUC no. 2018-0024). Blood (50 ml) was collected from the jugular vein using an 18-gauge needle and 60-ml syringe containing 5 ml of heparin (1000 U/ml; Sagent Pharmaceuticals), resulting in a final concentration of 100 U/ml of heparin. The blood was immediately stored on ice and transported to the laboratory for processing within 1 hour using Ficoll density gradient centrifugation (50). Briefly, freshly collected blood was diluted with equal volume of PBS containing 2% FBS (VWR Life Science Seradigm). A total of 20 ml of diluted blood was overlaid on 20-ml Ficoll-Paque PLUS (GE Healthcare, Chicago, IL) in a 50-ml conical tube. The Ficoll gradient centrifugation was performed at 400g for 30 min with low acceleration and no deceleration at room temperature. The PBMCs were collected from the plasma-Ficoll interface with a sterile Pasteur pipette and diluted 2:1 in PBS. After centrifugation at 300g for 5 min, the PBMCs pellet was resuspended in 10 ml of PBS and centrifuged at 300g for 5 min to wash the cells. The PBMCs were then cultured in RPMI supplemented with 1% GlutaMAX and 10% FBS.

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