In Vitro Stimulation of Mouse Splenocytes with Heat-Killed Listeria monocytogenes

Samenvatting

Source: Ahn, J. J., et al., Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses. J. Vis. Exp. (2016)

This video demonstrates the preparation of mouse splenocyte cultures from Listeria monocytogenes-infected mice for downstream cytokine analysis. It includes ex vivo bacteria-derived antigen stimulation and sample processing to study T cell responses.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Effects of Listeria monocytogenes on IFN-γ Responses by CD4⁺ and CD8⁺ Cells

NOTE: This procedure describes how to measure IFN-γ production by splenic CD4⁺ and CD8⁺ T effector cells harvested at the time of the peak of the adaptive immune response (~ 7 d post-infection) using two methods: (1) flow cytometry to measure IFN-γ (Interferon-gamma) by CD4⁺ and CD8⁺ cells by intracellular cytokine staini....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Brain Heart Infusion Broth, Modified	BD	299070	any brand should be appropriate
1x PBS	Sigma	D8537	any brand should be appropriate
RPMI 1640	Gibco	22400089	any brand should be appropriate
Fetal Bovine Serum	Gibco	12483	Before use, heat-inactivate at 56 °C for 30 min
GolgiStop Protein Transport Inhibitor (containing Monensin)	BD	554724	Use 4 μl in 6 ml cell culture
10x Perm/Wash buffer	BD	554723	Dilute 1x in ddH2O
50 ml Vented tubes for culture			Used for culturing the bacteria, any brand should be appropriate
1.5 ml Microcentrifuge tubes			any brand should be appropriate
Bacterial petri dishes			any brand should be appropriate
C57BL/6J Mouse	Jackson laboratories	Stock#000664	Order for arrival at 7 weeks
Bleach			For decontamination
70% Ethanol			For decontamination
Pipette Tips			any brand should be appropriate
Pipette			any brand should be appropriate
Pipette gun			any brand should be appropriate
Round bottomed plates			any brand should be appropriate