In vivo CTL assay

Friday, April 29, 2005

Description
This assay allows the detection of cytolytic activity specific for a peptide epitope in vivo in mice. One group of cells is labelled with a fluorescent dye and pulsed with a peptide epitope. The other group is labelled with a different dye and pulsed with an irrelevant peptide. They are mixed in a 1:1 ratio and injected i.v. into mice. A deviation from the 1:1 ratio when the mice are sampled indicates specific lysis of the cells with the relevant epitope on the surface. Normal spleen cells are used as target cells.

Procedure
1. Take spleen cells, lyse red blood cells and count
2. Incubate cells with the appropriate peptide at 1ug/ml in medium at a cell density of 20 million cells/ml. Incubate for 1 hour at 37oC.
3. Wash cells x1 with medium
4. Label one group of cells with CFSE and one with celltracker orange, according to protocols below:

CFSE labeling:
Wash cells x1 in HBSS
Resuspend in HBSS + 0.5uM CFSE at 2x107 cells/ml
Incubate for 10 minutes, room temperature in the dark
Wash cells x3 in medium
Count

Celltracker Orange labeling
Resuspend cells in CTM + 10uM celltracker orange at 2x107 cells /ml
Incubate for 30 minutes at 37oC (water bath)
Spin down cells and add warm CTM to 2x107 cells/ml
Incubate for a further 30 mins. at 37oC.
Wash cells x3 with 10mls CTM
Count

5. Resuspend both sets of cells at 100 million cells/ml then mix the desired pair of samples in a 1:1 ratio.
6. Inject 200ul i.v. into mice (i.e. 10 million of each group of cells). Save some cells to run on FACS to check cellular ratios.
7. Sample spleens or lymph nodes at the desired time post-infection (4-24 hours)
8. Lyse the red blood cells
9. Add 20ug/ml 7-aminoactinomycin D (7-AAD) in SWB to samples to be used in flow cytometry. Incubate 15minutes in the dark.
10. Run samples on flow cytometer: FL-1 is CFSE
FL-2 is Celltracker Orange
FL-3 is 7-AAD

Recipes
Specific lysis is calcuated by the formulas: ratio = (number CFSE/number CellTracker Orange);
percentage of specific lysis = (1 /(ratio of naive/ratio of
infected) X 100).

Supplies
CellTracker Orange and CFSE from Molecular Probes

Tips
Inclusion of 7-AAD allows dead or dying cells to be exclude from the analysis, giving cleaner results.

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In vivo CTL assay