Flow Cytometry: Innate lymphoid cells in lung tissue

Protocol ILC staining

Reagents needed:

• PBS + 0.5% BSA
• PBS + 0.5%BSA + 50mM EDTA
• Catalog number for FoxP3 Permeabilisation kit: 00-5521-00 – Thermo Fisher
• FoxP3 Fixation/Permeabilization working solution (1 ml needed per sample):
  • 1 part Foxp3 Fixation/Permeabilization concentrate
  • 3 parts Foxp3 Fixation/Permeabilization diluent
• 1X Permeabilization buffer (8.5ml needed per sample):
  • 1 part 10X Permeabilization buffer
  • 9 parts distilled water

Surface staining

1. Bring 2 up to 4 million cells into a tube
2. Centrifuge tube (1400 rpm, 5 min) and discard supernatans
3. Add 0.125ml FVD780 in 1 ml PBS
4. Incubate 30 minutes on room temperature in the dark
5. Wash with 2 ml PBS + 0.5% BSA
6. Add 1 µl of FC block (catalog number: 553142 – BD biosciences) to cells 
7. Incubate 10 minutes at 4°C in the dark
8. Add antibody mix (see table below)
9. Incubate 25-30 minutes at 4°C
10. Wash with 2 ml PBS + 0.5% BSA and discard supernatant

Intracellular staining

1. Add 1 ml of FoxP3 Fixation/Permeabilization working solution to tube and pipet up and down
2. Incubate for 50 minutes at 4°C in the dark
3. Add 2 ml of 1X Permeabilization buffer to tube and centrifuge (400g, 5 minutes) 
4. Discard supernatans
5. Resuspend pellet in residual volume of 1X Permeabilization buffer
6. Add RORyT-APC antibody to each tube
7. Incubate 30 minutes at 4°C in the dark
8. Add 2 ml of 1X Permeabilization buffer to tube and centrifuge (400g, 5 minutes) 
9. Discard supernatans
10. Add 2 ml of 1X Permeabilization buffer to tube and centrifuge (400g, 5 minutes) 
11. Discard supernatans
12. Fixate cells with 200 µl of 1% paraformaldehyde for 20 minutes at 4°C
13. Wash cells with 2 ml PBS+ 0.5% BSA
14. Dissolve pellet in 100 µl total volume – use PBS + 0.5% BSA + 50 mM EDTA

Antibodies ILC panel