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

Zusammenfassung

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.

Protokoll

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 staining, and (2) ELISA to measure total IFN-γ levels produced by splenocytes (includes all T cells). Procedures are performed within the Biological Safety Cabinet (BSC).

1. Infect mice by injecting i.p. with 2 x 10⁴ Colony-Forming Units (CFU) of the pathogen using procedures described in Procedure 4.
2. On day 7 post-infection, euthanize mice by CO₂ inhalation according to institutional guidelines.
3. Dissect the spleen and place it in a 15 ml conical tube containing sterile 1x PBS (Phosphate-Buffered Saline). Transport the tubes to the laboratory in a leak-proof container containing ice.
4. Process the spleens into a single cell suspension, lyse red blood cells as described in section 6, and then resuspend cells in complete RPMI (Roswell Park Memorial Institute) media containing 10% FCS (Fetal Calf Serum). Count cells using a hemocytometer.
5. Set up cultures for measuring IFN-γ responses. For this, dispense cells (4 x 10⁶ in 1 ml/well) into 24-well plates together with an equal number (4 x 10⁶ or 1 ml/well) of thawed heat-killed L. monocytogenes. Transfer cells to a 37 °C incubator.
6. After 20 hr of incubation, add 0.66 μl/ml of protein transport inhibitor to the wells and continue incubation.
7. Four hr later, transfer the plate to the BSC and collect 500 µl of culture supernatant and freeze (at -80 °C) for the later measurement of IFN-γ levels using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Then, collect cells into a sterile 15 ml tube. Wash wells with 1x PBS and pool this wash together with the collected cells.
8. Conduct cell-surface staining and intracellular staining for IFN-γ on CD4⁺ and CD8⁺ cells. Proceed to flow cytometer acquisition (collecting 200,000 events/sample) and analyze data using flow cytometry analysis software.

Materialien

Liste der in diesem Artikel verwendeten Materialien

Name	Unternehmen	Katalognummer	Kommentare
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