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…
Divulgaciones
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 |
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A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation. J. Vis. Exp. (Pending Publication), e21919, doi: (2024).