A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

Published: January 31, 2024

Abstract

Source: Liaqat, F., et al., Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells. J. Vis. Exp. (195), (2023).

This video demonstrates a monocyte-derived dendritic cell, MoDC-based assay to measure the immunogenicity of commercially available vaccines. MoDC assays investigate dendritic cell responses to antigens, pathogens, or treatments and can provide insights into immune responses, vaccine development, and immunotherapy research.

Protocol

1. Production of Naïve MoDCs NOTE: Whole blood samples were obtained from a single pathogen-free calf by jugular vein puncture with heparinized vacutainers (eight 9 mL blood tubes were used for this study). Transport the blood in an ice box. Store the samples at 2-4 °C for later use, or process them immediately. Keep the blood rotating to avoid blood clotting. Sterilize the vacutainers with 70% ethanol. All the following experiments were performed with…

Açıklamalar

The authors have nothing to disclose.

Materials

ACK Lysing Buffer Gibco, Thermo Fisher A1049201 Ammonium-Chloride-Potassium buffer for lysis of residual RBCs in harvested PBMC Fraction
Bovine Dendritic Cell Growth Kit Bio-Rad, UK PBP015KZZ Cytokine cocktail composed of recombinant bovine IL-4 and GMCSF
Bovine IFN-γ ELISA Kit Bio-Rad MCA5638KZZ Kit use for measuring IFN-γ expression in culture supernatant
Nobivac Rabies MSD Animal Health, UK 1 µL/mL of cell cultured inactivated vaccine containing> 2 I.U./mL Rabies virus strain
Optical seals Bi0-Rad TCS0803 0.2 mL flat PCR tube 8-cap strips, optical, ultraclear, compatible for qPCR machine
Phosphate Buffer Saline (PBS) Gibco, Thermo Fisher 10010023 pH 7.4, 1x concentration
FlowTubes/ FACS (Fluorescenceactivated single-cell sorting) Tube Falcon Corning 352235 5 mL, sterial, round bottom polystyrene test tube with cell strainer snap cap, use in flow cytometry analysis
Human CD14 MicroBeads Miltenyi Bioteck, Germany 130-050-201 2 mL microbeads conjugated to monoclonal anti-human CD14 antibody isotype IgG2a, used for selection of bovine monocytes from PBMCs
Purified Anti-human Ki-67 antibody Biolegend, USA 350501 Monoclonal antibody, cross reactive with cow, clone ki-67
RPMI 1640 Medium Sigma Aldrich R8758 Cell culture media with L-glutamine and sodium bicarbonate
Tissue Culture Test plate 24 TPP, Switzerland 92024 24 well plate, sterilized by radiation , growth enhanced treated, volume 3.18 mL

Etiketler

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Bu Makaleden Alıntı Yapın
A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation. J. Vis. Exp. (Pending Publication), e21919, doi: (2024).

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