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

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 one biological sample and six technical replicates.

1. Density gradient centrifugation to isolate PBMCs from the heparinized blood
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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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