Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Abstract

Source: Mazet, J. M., et al. Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection. J. Vis. Exp. (2019).

This video demonstrates an imaging method to visualize in situ interferon-gamma (IFN-γ) secretion in the mouse spleen. Following bacterial infection and the induction of IFN-γ accumulation inside T cells and natural killer cells, the spleen is harvested from the mouse. Upon obtaining tissue sections, immunostaining is performed to visualize IFN-γ inside the cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Adoptive Transfer of Antigenic-specific CD8+ T Cells in Mice

1. Isolate ovalbumin (OVA)-specific CD8⁺ T cells (OTI) expressing green fluorescent protein (OTI-GFP) or red fluorescent protein (OTI-RFP) from lymph node suspension of T-cell receptor transgenic mice using a mouse CD8⁺ T cell isolation kit as per manufacture instructions. Prepare the cell suspension by smashing lymph nodes using a syringe plunger, as previously described.
2. Trans....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Brefeldin A	Cambridge bioscience	CAY11861
Paraformaldehyde	Agar scientific	R1018
L-Lysin dihydrochloride	Sigma lifescience	L5751
Sodium meta-periodate	Thermo Scientific	20504
D(+)-saccharose	VWR Chemicals	27480.294
Precision wipes paper Kimtech science	Kimberly-Clark Professional	75512
O.C.T. compound, mounting medium for cryotomy	VWR Chemicals	361603E
Fc block, purified anti-mouse CD16/32, clone 93	Biolegend	101302	Antibody clone and Concentration used: 2.5 mg/ml
Microscope slides - Superfrost Plus	VWR Chemicals	631-0108
anti-CD169 - AF647	Biolegend	142407	Antibody clone and Concentration used: clone 3D6.112 1.6 mg/ml
			Excitation wavelength: 650
			Emission wavelength: 65
anti-F4/80 - APC	Biolegend	123115	Antibody clone and Concentration used: clone BM8 2.5 mg/ml
			Excitation wavelength: 650
			Emission wavelength: 660
anti-B220 - PB	Biolegend	103230	Antibody clone and Concentration used: clone RA3-6B2 1.6 mg/ml
			Excitation wavelength: 410
			Emission wavelength: 455
anti-IFNg - biotin	Biolegend	505804	Antibody clone and Concentration used: clone XMG1.2 5 mg/ml
anti-IFNg - BV421	Biolegend	505829	Antibody clone and Concentration used: clone XMG1.2 5 mg/ml
			Excitation wavelength: 405
			Emission wavelength: 436
anti-Nkp46/NCRI	R&D Systems	AF2225	Antibody clone and Concentration used: goat 2.5 mg/ml
anti-goat IgG-FITC	Novusbio	NPp 1-74814	Antibody clone and Concentration used: 1 mg/ml
			Excitation wavelength: 490
			Emission wavelength: 525
Streptavidin - PE	Biolegend	405203	Antibody clone and Concentration used: 2.5 mg/ml
			Excitation wavelength: 565
			Emission wavelength: 578
streptavidin - FITC	Biolegend	405201	Antibody clone and Concentration used: 2.5 mg/ml
			Excitation wavelength: 490
			Emission wavelength: 525
Fluoromount G	SouthernBiotech	0100-01
Cover glasses 22x40mm	Menzel-Glazer	12352128
Liquid blocker super PAP PEN mini	Axxora	CAC-DAI-PAP-S-M
Imaris - Microscopy Image Analysis Software	Bitplane
Confocal microscope - Olympus FV1200 Laser scanning microscope	Olympus
Cryostat - CM 1900 UV	Leica
Base mould disposable	Fisher Scientific UK Ltd	11670990
PBS 1X	Life Technologies Ltd	20012068
BHI Broth	VWR Brand	303415ZA
GFP			Excitation wavelength: 484
			Emission wavelength: 507
RFP			Excitation wavelength: 558
			Emission wavelength: 583
Insulin syringe, with needle, 29G	VWR International	BDAM324824
C57BL/6 wild type mice	Charles River