MAIT Cell Intracellular Cytokine Staining

Prewarm collagenase media and shaking incubator to 37°C.

Mice should be euthanized (e.g., using a rising concentration of CO₂ with a second method to confirm death).

Open the diaphragm by cutting the rib cage to expose both the heart and lungs. Gently perfuse the right ventricle with 8mL–10mL to remove circulating blood. Perfuse using a 10-mL syringe and a 26-G needle. Proper perfusion will result in lung inflation and a color change to pink/ white.

Remove lungs ( see Note 18 ) using scissors to cut through the hilum and place into a 24-well plate containing ice-cold RPMI to transfer organs to the laboratory.

Chop lungs into very small pieces ( see Note 9 ).

Place lung tissue into a 1-mL Eppendorf tube containing 0.5 mL/lung of pre-warmed collagenase/DNase medium. This should also contain, 0.5µL (final concentration 3.0μg/mL).

Incubate tubes on their sides in a shaking incubator at 37°C, at 100rpm,0h 0m 0s–180rpm,0h 0m 0s, for 1h 30m 0s.

After 1h 30m 0s pour digested tissue through a 70-μm cell strainer and force through into Petri dish with the plunger from a 1-mL syringe. Rinse residual cells into a total of 10mL in 10 mL falcon tubes at Room temperature. Centrifuge at 400x g.

Resuspend in 2mL per lung ( see Note 19 ) of pre-warmed TAC lysis buffer at 37°C. Vortex well, then place in a pre-warmed water bath at 37°C. After 0h 5m 0s neutralize by adding an equal volume of FACS buffer. Centrifuge at 400x g.

Numbers of lung cells can be estimated using a hemocytometer or spectrophotometer ( see Notes 20 and 21 ).

Transfer 100µL containing 0.5–1 million cells to a 96-well U- or V-bottom plate format or into FACS tubes for staining, passing them through a 40-μm mesh ( see Note 22 ).

(a) 1 × 100 μL into a plate for surface stain ( steps 16–22 ).

(b) 2 × 100 μL (unstimulated and stimulated) to a second plate ( see Note 23 ), and include a no Brefeldin control ( steps 12 , 13 , and 21–30 ).

Keep the cells for the surface stain On ice, while setting up PMA/Ionomycin stimulation to induce production of cytokines of interest.

(a) PMA final concentration: 20ng/mL.

(b) Ionomycin 1μg/mL.

(c) 1000x stock Brefeldin A (final concentration 3.0μg/mL).

Incubate for 3h 0m 0s at 37°C with 5%.

During stimulation phase perform surface staining for extracellular markers ( see Note 24 ).

If performing Zombie Yellow vital staining wash cells with 1mL–2mL. Centrifuge at 400x g (or if using plate format wash twice with 200µL centrifuging for 0h 2m 0s at 400x g,0h 0m 0s). Resuspend in 20µL + 0.4µL for 0h 15m 0s.

Add 20µL containing 0.2µL to block non-specific binding. Incubate for 0h 15m 0s dark, Room temperature.

Add surface cocktail (Table 2) using a cocktail made up in 10µL. Pipette carefully to mix. Stain for 0h 20m 0s–0h 30m 0s at Room temperature.

For single color controls use splenocytes or compensation beads.

Wash cells twice with 2mL, centrifuging at 400x g (or if using plate format wash three times with 200µL centrifuging for 0h 2m 0s at 400x g,0h 0m 0s).

Resuspend cells in 100µL ( see Note 25 ). To enable estimation of absolute cell numbers, add a known number of calibration beads.

(a) Vortex calibration beads hard. Dilute (1:10) counting beads in PBS before using. To each sample 25µL was added, and an additional 10µL were saved to be counted with a hemocytometer, giving a count of X in a large square, i.e., X × 10⁴ beads/mL (which is X x 10 beads/μL, or X × 10 × 25 beads/sample). Typically add a total of 25,000 beads per sample.

(b) When samples have been acquired on flow cytometer, these calibration beads can be detected using their FSC/SSC profile and the absolute number of cells of interest can be estimated using the following approach. Total number of MAIT cells per sample = Number of MAIT cells counted on flow cytometer × Number of beads added/Number of beads counted/proportion of total lung cell suspension actually used for staining.

After 3-h stimulation, continue processing the cells for intracellular staining. Resuspend into FACS tube with +1mL. Centrifuge at 400x g. (Alternatively, if in 96-well format resuspend in 100µL, centrifuge at 400x g and repeat.)

Resuspend in 20µL with 0.4µL for 0h 15m 0s.

Add 20µL containing 0.2µL to block non-specific tetramer staining. Incubate for 0h 15m 0s dark, Room temperature.

Add surface cocktail (Table 3) using a cocktail made up in 10µL. Pipette carefully to mix. Stain for 0h 20m 0s–0h 30m 0s at Room temperature.

For single color controls use splenocytes or compensation beads. These may be available from being made up earlier in the protocol.

Wash cells once with 1mL, centrifuging at 400x g (or if using plate format wash twice with 200µL centrifuging for 0h 2m 0s at 400x g,0h 0m 0s).

Resuspend in 200µL and incubate for 0h 30m 0s On ice.

Wash with 2mL. Centrifuge at 400x g (or if using plate format wash twice with 200µL centrifuging for 0h 2m 0s at 400x g,0h 0m 0s).

Resuspend in 50µL containing intracellular cocktail (Table 4) and pipette carefully to mix. Incubate for 0h 45m 0s or leave to stain 0h 45m 0s.

Wash cells with 2mL (or if using plate format wash twice with 200µL centrifuging for 0h 2m 0s at 400x g,0h 0m 0s). Resuspend cells in 100µL. If cells are in plate format use a multichannel pipette to transfer them to 1.2 mL “bullet” cluster tubes for acquisition, or use a plate reader attachment with the cytometer.

Analyze cells on flow cytometer.