24 well flat bottomed culture plates

Heparinized peripheral venous blood samples from dewormed healthy goats were collected and cultured as the procedure described by Yanming et al. [22 (link)]. PBMCs were separated by standard Ficoll-hypaque (GE Healthcare, Munich, USA) gradient centrifugation method [23 (link)]. After washing twice with Ca²⁺/Mg²⁺-free PBS (pH 7.4), the trypan blue exclusion test conducted for cell viability was more than 95% in all experiments. PBMCs were adjusted to the required density in cell culture medium (RPMI 1640 or DMEM), containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (GIBCO, Paisley, UK). For functional analysis, PBMCs were cultured in 24-well flat-bottomed culture plates (Costar, Cambridge, MA, USA) with varying concentrations of rHc-AK at 37 °C in 5% CO₂.

Whole blood was taken by venipuncture of the jugular vein, drained into 10 mL vacutainers coated with ethylenediaminetetraacetic acid (EDTA) and brought to laboratory for PBMCs isolation and culture. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-hypaque (GE Healthcare, Madison, WI, USA) density gradient centrifugation protocol [27 (link)]. Following a washing step (three times) with PBS (Ca²⁺/Mg²⁺-free pH 7.4) cell viability was assessed by a Trypan Blue exclusion test for consistency >95%. Cells with a final density at 1 × 10⁶ cells/mL were incubated with Roswell Park Memorial Institute (RPMI) 1640 culture media containing 10% heat-inactivated fetal bovine serum (FBS) (ThermoFisher, Waltham, MA, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (GIBCO, Grand Island, New York, USA). For functional study, PBMCs were cultured in 24-well flat-bottomed culture plates (Costar, Cambridge, MA, USA) with varying concentrations of rHc-TpMy at 37 °C and 5% CO₂ for different time periods according to the test required.