Ficoll paque premium density gradient medium

The buffy coat was diluted 1:1 with sterile phosphate‐buffered saline (PBS), and peripheral blood mononuclear cell (PBMC) isolation was performed using Ficoll^®‐Paque PREMIUM density gradient medium (GE Healthcare, Little Chalfont, UK) according to the manufacturer's recommendations. Isolated PBMCs were cultured in T75 flasks for monocyte selection and stimulated to monocyte‐derived dendritic cells with recombinant human IL‐4 and granulocyte‐macrophage colony‐stimulating factor (Luplertlop et al., 2006). An African green monkey kidney cell line (Vero) was kindly provided by Dr A. Thontiravong of the Faculty of Veterinary Medicine, Chulalongkorn University, Thailand. The cells were cultured in RPMI 1640 media (Gibco, Waltham, MA). Lilly Laboratories Cell—Monkey Kidney 2 (LLC‐MK2) cells were cultured in Dulbecco's modified Eagle's medium (Gibco). All cell cultures were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco) and incubated at 37°C, 5% CO₂ in a humidified incubator.

Rabbit PBMCs were isolated from 5 mL blood collected from the central artery of the rabbit ear before infection and at various time-points post-infection. Immediately after euthanasia, single-cell suspensions were prepared from popliteal lymph nodes and spleen as follows. Tissues were gently disrupted with scissors in sterile RPMI-1640 medium and passed through a 70 μm cell-strainer using a 1-mL syringe plunger. Mononuclear leukocyte suspensions were prepared from peripheral blood and tissue samples using Ficoll-Paque Premium density gradient medium (GE Healthcare). Each 5 mL single-cell suspension was diluted 1:1 in sterile PBS, overlaid onto a 5 mL Ficoll-Paque density cushion, and centrifuged (1825×g) for 20 min at room temperature. Mononuclear leukocytes at the interface were collected and washed in ice-cold PBS before further analysis. Bovine blood was obtained from a healthy donor of CARE-FEPEX ("Cellule d’Appui à la Recherche et à l’Enseignement–Ferme Pédagogique et Experimentale", Dr L. Martinelle) and PBMCs isolated on Ficoll-Paque density gradient using SepMate isolation tubes (STEMCELL technologies).