Ficoll hypaque

PBMCs were isolated by Ficoll-Hypaque (1.077 g/cm³, Paneco, Russia) density gradient centrifugation (400g, 30 min). B cells were purified by magnetic separation using the CD19-MicroBeads (Miltenyi Biotec, Germany). Cells were cultured in full RPMI-1640 medium supplemented with 2mМ glutamine (Paneco), 20% FBS (Biosera, France), 1 mМ sodium pyruvate, 10 mМ HEPES, 100 U/ml penicillin, and 100 mcg/ml streptomycin (all Paneco).

Monocyte-derived dendritic cells (DC) were generated as described previously [10 (link)]. Briefly, fresh peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Ficoll-Hypaque (PanEco, Moscow, Russia) gradient centrifugation and were then allowed to adhere to 12-well culture plates for 1 h. Nonadherent cells were collected and centrifuged, and cell pellets were mixed with autologous serum containing 10% DMSO and stored in liquid nitrogen. Cryopreserved, nonadherent PBMCs, which also are considered as peripheral blood lymphocytes, were later used as a source of effector cells (cytotoxic T lymphocytes, CTL) for cytotoxicity assays. The adherent cell fraction was cultured in RPMI-1640 (Gibco) supplemented with 10% FBS, streptomycin-penicillin, and glutamine in the presence of 0.075 μg/mL granulocyte macrophage colony-stimulating factor (Neostim, 1.67 × 10⁶ ME, FDS FARMA, UK) and 1000 U/mL interleukin-4 (Sigma-Aldrich). After 6 d in culture, SANTAVAC (0.5 mL) or FAA (0.5 mL) were added to each well of a 12-well culture plate with immature DC (3 × 10⁵ cells/well in 1 mL of culture medium) and DC were matured with 1000 U/mL tumor necrosis factor-α (Sigma-Aldrich) for 48 h. Matured, SANTAVAC-loaded or FAA-loaded DC were then used to stimulate CTL.

PMA, A23187, Triton X-100, and fetal calf serum (FCS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Wheat germ agglutinin (WGA)-AlexaFluor 594 conjugate, Phalloidin-AlexaFlour 488 conjugate, and DAPI were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Dextran T-500 was obtained from Pharmacosmos (Holbæk, Denmark). RPMI1640, Hoechst 33342, Ficoll-Hypaque, L-glutamine, and HEPES were obtained from PanEco (Moscow, Russia). Coverslips and 24-well plates were from SPL Co., Ltd. (Pyeongtaek, Republic of Korea). FITC-labeled monoclonal mouse anti-human MPO priming antibodies were purchased from Invitrogen (Waltham, MA, USA).