TITLE: MAIT Cell Intracellular Cytokine Staining
AUTHORS: Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Alexandra J. Corbett, Zhenjun Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 3.5 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract: </span><span>MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.</span></div></div>

[STEPS]
?. [To analyze MAIT cell frequencies and function during viral infection.]
Prewarm collagenase media and shaking incubator to .
37 °C
?. [To analyze MAIT cell frequencies and function during viral infection.]
Mice should be euthanized (e.g., using a rising concentration of CO2 with a second method to confirm death).
?. [To analyze MAIT cell frequencies and function during viral infection.]
Open the diaphragm by cutting the rib cage to expose both the heart and lungs. Gently perfuse the right ventricle with – 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.
8 mL
[ice-cold RPMI]
?. [To analyze MAIT cell frequencies and function during viral infection.]
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.
?. [To analyze MAIT cell frequencies and function during viral infection.]
Chop lungs into very small pieces (see Note 9).
?. [To analyze MAIT cell frequencies and function during viral infection.]
Place lung tissue into a 1-mL Eppendorf tube containing 0.5 mL/lung of pre-warmed collagenase/DNase medium. This should also contain,  (final concentration ).
[Brefeldin A (1:1000)]
?. [To analyze MAIT cell frequencies and function during viral infection.]
Incubate tubes on their sides in a shaking incubator at , at –, for .
37 °C
Centrifuge: 100 33
Centrifuge: 180 33
?. [To analyze MAIT cell frequencies and function during viral infection.]
After  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  in 10 mL falcon tubes at . Centrifuge at .
[FACS wash]
0 Room temperature
Centrifuge: 400 34, 5 min
?. [To analyze MAIT cell frequencies and function during viral infection.]
Resuspend in  per lung (see Note 19) of pre-warmed TAC lysis buffer at . Vortex well, then place in a pre-warmed water bath at . After  neutralize by adding an equal volume of FACS buffer. Centrifuge at .
2 mL
37 °C
37 °C
Centrifuge: 400 34, 5 min
?. [To analyze MAIT cell frequencies and function during viral infection.]
Numbers of lung cells can be estimated using a hemocytometer or spectrophotometer (see Notes 20 and 21).
?. [To analyze MAIT cell frequencies and function during viral infection.]
Transfer  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).
100 µl
?. [In vitro stimulation phase:]
Keep the cells for the surface stain , while setting up PMA/Ionomycin stimulation to induce production of cytokines of interest.(a) PMA final concentration: .(b) Ionomycin .(c) 1000x stock Brefeldin A (final concentration ).
on ice
?. [In vitro stimulation phase:]
Incubate for  at  with .
37 °C
[CO2]
?. [Surface staining:]
During stimulation phase perform surface staining for extracellular markers (see Note 24).
?. [Surface staining:]
If performing Zombie Yellow vital staining wash cells with –. Centrifuge at  (or if using plate format wash twice with  centrifuging for  at ). Resuspend in  +  for .
1 mL
[PBS]
Centrifuge: 400 34, 5 min
[FACS buffer]
Centrifuge: 400 34
[ PBS]
[ZombieYellow]
?. [Surface staining:]
Add  containing  to block non-specific binding. Incubate for  dark, .
[2.4G2 (anti-CD16/32)]
[MR1-6-FP tetramer (no fluorochrome conjugate)]
0 Room temperature
?. [Surface staining:]
Add surface cocktail (Table 2) using a cocktail made up in . Pipette carefully to mix. Stain for – at .
[FACS buffer]
0 Room temperature
?. [Surface staining:]
For single color controls use splenocytes or compensation beads.
?. [Surface staining:]
Wash cells twice with , centrifuging at  (or if using plate format wash three times with  centrifuging for  at ).
[FACS buffer]
Centrifuge: 400 34, 5 min
[FACS buffer]
Centrifuge: 400 34
?. [Surface staining:]
Resuspend cells in  (see Note 25). To enable estimation of absolute cell numbers, add a known number of calibration beads.
[FACS buffer]
?. [Surface staining:]
(a)   Vortex calibration beads hard. Dilute (1:10) counting beads in PBS before using. To each sample  was added, and an additional  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.
[of these diluted beads]
[of beads]
?. [Surface staining:]
(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.
?. [Intracellular staining:]
After 3-h stimulation, continue processing the cells for intracellular staining. Resuspend into FACS tube with +. Centrifuge at . (Alternatively, if in 96-well format resuspend in , centrifuge at  and repeat.)
[PBS]
Centrifuge: 400 34, 5 min
[PBS]
Centrifuge: 400 34, 2 min
?. [Intracellular staining:]
Resuspend in  with  for .
[PBS]
[Zombie Yellow]
?. [Intracellular staining:]
Add  containing  to block non-specific tetramer staining. Incubate for  dark, .
[2.4G2 (anti CD16/32) SN]
[unlabeled MR1-6-FP tetramer]
0 Room temperature
?. [Intracellular staining:]
Add surface cocktail (Table 3) using a cocktail made up in . Pipette carefully to mix. Stain for – at .
[FACS buffer]
0 Room temperature
?. [Intracellular staining:]
For single color controls use splenocytes or compensation beads. These may be available from being made up earlier in the protocol.
?. [Intracellular staining:]
Wash cells once with , centrifuging at  (or if using plate format wash twice with  centrifuging for  at ).
[FACS buffer]
Centrifuge: 400 34, 5 min
[FACS buffer]
Centrifuge: 400 34
?. [Intracellular staining:]
Resuspend in  and incubate for .
[commercially available Fixation/Permeabilizationsolution]
on ice
?. [Intracellular staining:]
Wash with . Centrifuge at  (or if using plate format wash twice with  centrifuging for  at ).
[Perm Wash (diluted 1:9 with FACS buffer)]
Centrifuge: 400 34, 5 min
[Perm Wash]
Centrifuge: 400 34
?. [Intracellular staining:]
Resuspend in  containing intracellular cocktail (Table 4) and pipette carefully to mix. Incubate for  or leave to stain .
[Perm Wash]
?. [Intracellular staining:]
Wash cells with  (or if using plate format wash twice with  centrifuging for  at ). Resuspend cells in . 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.
[PermWash]
[Perm Wash]
Centrifuge: 400 34
[FACS wash]
?. [Intracellular staining:]
Analyze cells on flow cytometer.