An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Published: March 29, 2024

Abstract

Source: Lirussi, D. et al., Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development. J. Vis. Exp. (2018)

The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. All mice used in this study were from the C57BL/6 background. All the animals were kept under pathogen-free conditions. 1. Carboxyfluorescein succinimidyl ester (CFSE) Staining of OT-I T Cells and Adoptive Transfer NOTE: OT-I mice are transgenically generated anima…

Representative Results

Figure 1: The assay timeline. The assay timeline represents the initial OT-I T cell transfer at day 0, the s.c. vaccination at day 1, and the sampling spleen and draining of lymph nodes 2 days later. Figure 2: Flow diagram of the gating strategy fo…

Açıklamalar

The authors have nothing to disclose.

Materials

BD LSR Fortessa Cell Analyzer BD Special Order Flow Cytometer
CFSE Molecular Probes C34554 Proliferation Dye
MojoSort Mouse CD8 T Cell Isolation Kit Biolegend 480007 Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.
LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation Molecular Probes L23105 Dead Cell Marker
CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7 eBioscience 25-0900-82 antibody
APC anti-mouse CD8a Antibody BioLegend 100712 antibody
BV421 Rat Anti-Mouse CD4 BD 740007 antibody
Z2 coulter Particle count and Size Analyzer Beckman Coulter 9914591DA Cell counter. Z2 Automated particle/cell counter
EndoGrade Ovalbumin (10 mg) Hyglos(Germany) 321000 Ovalbumin endotoxin free tested.
Cell Strainer 100µm nylon Corning 352360 Cell strainer (100 µm pore mesh cups).
Sample Vials Beckman Coulter 899366014 Sample vials for Z2 automated counter
C57BL/6 mice (CD90.2) Harlan (Rossdorf, Germany) Company is now Envigo
OT-I (C57BL/6 background, CD90.1) Harlan (Rossdorf, Germany) Inbreed at our animal facility. Company from where adquired is now Envigo
FACS tubes Fischer (Corning) 14-959-5 Corning Falcon Round-Bottom Polystyrene Tubes
Falcon 15 mL tubes Fischer (Corning) 05-527-90 Falcon 15mL Conical Centrifuge Tubes
PBS (500 mL) Fischer (Gibco) 20-012-027 Gibco PBS (Phosphate Buffered Saline), pH 7.2
Red lamp (heating lamp) Dirk Rossmann GmbH (Germany) 405096 Heating infrred lamp (100 wats)
IsoFlo (Isoflurane) Abbott Laboratories (USA) 5260.04-05. Isoflurane anesthesic (250 mL flask).
Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber Parkland Scientific V3000PK Isoflurane anesthesia machine.
RPMI 1640 medium Gibco (distributed by ThermoFischer) 11-875-093 Base medium with Glutamine (500 mL)
Pen-Strept antibiotic solution (Gibco) Gibco (distributed by ThermoFischer) 15-140-148 Gibco Penicillin-Streptomycin (10,000 U/mL)
Fetal Bobine Serum (Gibco) Gibco (distributed by ThermoFischer) 10082147 Fetal Bovine Serum, certified, heat inactivated, US origin
ACK Lysing Buffer (100 ml) Gibco (distributed by ThermoFischer) A1049201 Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also
Plastic Petri Dishes Nunc (distributed by ThermoFischer) 150340 60 x 15mm Plastic Petri Dish, Non-treated
Cell Clump Filter CellTrics (Sysmex) 04-004-2317 CellTrics® 50 μm, sterile

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An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation. J. Vis. Exp. (Pending Publication), e22012, doi: (2024).

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