Isolation and labeling of CTL (day 1)
Isolate spleens from CTL-donor mice and generate a single cell suspension by extruding the spleen through a steel sieve with the plunger of a 2 ml syringe and filtering through a Nylon sieve (100 µm).
Centrifuge cell suspension at 300 g for 6 mins, 4°C.
Resuspend cells in 600 µl of MACS buffer, add 20 µl of alpha murine CD8 MicroBeads per spleen and incubate for 15 mins at 4°C.
Centrifuge at 300 g for 6 mins, 4°C.
In the meantime, embed MACS columns (1 column per 2-3 spleens) in magnetic field and equilibrate with 3 ml of MACS buffer.
Resuspend cells in 3 ml of cold MACS buffer, then pipette filtered cell suspension on column and wash with 3 ml MACS buffer.
Remove MACS columns from magnetic field and rinse with 4 ml MACS buffer. Capture passage (= positive fraction of cells) in a 15 ml tube.
Centrifuge at 300 g for 6 mins, 4°C.
Resuspend cells in 5 ml of PBS in a 15 ml tube and add 2.5 µl of FarRed staining solution into the lid of the tube, and invert tube carefully. Incubate for 20 mins at 37 °C.
CRITICAL STEP Due to potential cytotoxicity of the staining reagent, do not exceed incubation time of 20 mins.
Stop staining reaction by adding 7 ml of ice-cold PBS and centrifuge at 300 g for 6 mins, 4 °C.
Resuspend cells in PBS and adjust the cell number to 12.5 x 106 cells per ml of PBS. Inject 200 µl (2.5 x 106 cells) intravenously into mice of favored genetic background.
Administration of antigen and adjuvant (day 2)
- Inject antigen with or without adjuvant intravenously into mice 16 hrs after adoptive T cell transfer. Injection of antigen alone should be included as a control.
Preparation of cryosections (day 2)
- After 10 hrs, isolate spleens from injected mice, rinse them briefly in ice-cold PBS, and embed them directly in Tissue-Tek®.
- Store at -20°C over night.
PAUSE POINT Tissue blocks can be stored at -20°C for up to a week or for longer time after transfer to -80°C.
Immunofluorescence staining and quantification of cell numbers (day 3)
Prepare 5 µm cryosections from frozen tissue blocks by using a microtome/cryostat at a working temperature of -18°C.
CRITICAL STEP Discard at least two sections between individual slides to avoid staining of the same cells in different sections.
Transfer sections on microscope slides for further treatment.
PAUSE POINT Dry sections for several hours.
CAUTION Avoid extended exposure to light.
Fix sections with iced acetone for 10 mins, then air-dry them for some minutes.
CRITICAL STEP Do not exceed fixation time of 10 mins. Otherwise, tissue integrity might be lost. The fixation might be performed at 4°C.
Apply blocking solution (1% BSA in PBS) to samples for 1 h at room temperature.
Stain sections with alpha murine B220-PE antibody for 1 h at room temperature, then wash twice with PBS.
Cover sections with mounting medium (Immu-Mount) and cover slides.
PAUSE POINT Slices can be stored at room temperature for up to one week.
View sections and capture images with a fluorescence microscope. Determine number of labeled cells with appropriate software. We used an Olympus IX71, employing a 10x objective. T cell numbers were counted (utilizing the "Touch count" mode) and areas analyzed ("Closed polygon") with Cell F software (Olympus). Furthermore, if distinct labels for multiple cell types are used, intercellular contacts can be enumerated.