TITLE: Mitochondrial staining of NK cells by flow cytometry
AUTHORS: Philippa R Kennedy

[DESCRIPTION]
Mitotracker Green (Thermo Fisher Scientific, cat. M7514) stains mitochondrial membranes, so gives a quick measure of mitochondrial mass per cell. However, it is not always as sensitive as quantifying mitochondrial mass by microscopy and staining intensity can be affected by mitochondrial swelling or reactive oxygen species.

Mitochondrial membrane potential assay kit (Cell Signaling Technology cat. 13296S) gives a fluorescent indication of the charge across mitochondrial membranes and thus the health of the mitochondria. A membrane charge uncoupler is included in the kit that depolarizes mitochondrial membranes and is thus a useful negative control.

MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific cat. M36008) detects reactive oxygen species within the cell. To see how much reactive oxygen species are generated in the face of oxidative stress, the cells can be pre-incubated with hydrogen peroxide and then stained with MitSOX indicator.

TO-PRO-3 (Thermo Fisher Scientific, cat. T3605) is a non-membrane permeable DNA-dye to discriminate live cells. Staining for CD56+ CD3- cells allows analysis of NK cells, not any contaminating cells. 

These stains are performed on live cells and analyzed without fixation.

[STEPS]
SECTION: Mitotracker and TMRM combined staining (keep on ice)
1. Resuspend 1x10^6 cells in 850 μL RPMI medium (no serum) for all tubes. Keep tubes on ice
SECTION: Mitotracker and TMRM combined staining (keep on ice)
2. Before diluting reagents, set up the CCCP negative controls:
Add 1 μL CCCP to tubes containing 1x10^6  cells in 850 μL RPMI
Incubate at 37°C (no CO2) for 20 min.
SECTION: Mitotracker and TMRM combined staining (keep on ice)
3. Prepare staining reagents and keep on ice:
TMRM: 
Stock (can be freeze-thawed): prepare by adding 55 μL DMSO to lyophilized TMRM
Working dilution: 2 μL stock into 1 mL RPMI

Mitotracker Green: 
Stock (can be freeze-thawed): Add 74.48 μL DMSO to tube contents to obtain 1 mM Mitotracker Green. 
Working dilution 100 nM in RPMI (i.e. 1:100 to get 10 μM stock; then 1:100 to get 100 nM solution)
SECTION: Mitotracker and TMRM combined staining (keep on ice)
4. After CCCP incubation, add TMRM and Mitotracker Green to ALL tubes (including CCCP controls):
100 μL TMRM
50 μL Mitotracker Green (from 100nM working solution)
Incubate at 37°C (no CO2) for 20 min.
SECTION: Mitotracker and TMRM combined staining (keep on ice)
5. Wash with flow buffer (1% human AB serum, 0.5 mM EDTA in PBS).
If staining in a 96 well plate, wash 3 times with 200 μl flow buffer. 
If staining in a tube, wash once with 3 mL flow buffer.
Keep on ice until surface staining
SECTION: MitoSOX staining (keep on ice)
7. Spin down 500,000 cells per tube and remove supernatant (400g for 5 min)
SECTION: MitoSOX staining (keep on ice)
8. Dilute reagents:
Stock (can be freeze-thawed if necessary): Dissolve MitoSOX reagent in 13 μL DMSO to make 5 mM solution
Dilute 5mM solution 1:1000 in Hanks' balanced salt solution for 5 μM solution
For DMSO control, dilute DMSO 1:1000 in Hanks' balanced salt solution.
SECTION: MitoSOX staining (keep on ice)
9. Add 200 μL working solution to each tube
SECTION: MitoSOX staining (keep on ice)
10. Incubate cells at 37°C for 15 min.
SECTION: MitoSOX staining (keep on ice)
11. Wash cells with flow buffer
If staining in a 96 well plate, wash 3 times with 200 μl flow buffer. 
If staining in a tube, wash once with 3mL flow buffer.
SECTION: Stain for live NK cells
12. Prepare stain mix with anti-CD3 and anti-CD56
anti-CD3 BV785 – 5 μL/sample
anti-CD56 PE-Cy7 – 5 μL/sample
Flow buffer – 90 μL/sample
SECTION: Stain for live NK cells
13. After spinning down cells and completing washes (see above), add 100 μL stain/tube.
SECTION: Stain for live NK cells
14. Incubate at 4°C for 15 min.
SECTION: Stain for live NK cells
15. Wash twice with flow buffer
SECTION: Stain for live NK cells
16. Dilute TO-PRO-3 dye (1:10,000) in flow buffer.
SECTION: Stain for live NK cells
17. Add 300 μL flow buffer per tube
SECTION: Stain for live NK cells
18. Add 200 μL diluted TO-PRO-3 per tube
SECTION: Stain for live NK cells
19. Run immediately on flow cytometer (LSRII, BD Biosciences)
SECTION: (Optional) Stress mitochondria with hydrogen peroxide
6. Prior to harvesting cells for mitoSOX staining, they can be stressed with hydrogen peroxide: 
Resuspend 500,000 cells/condition in fresh media. 
Add a titration of H2O2 e.g. 0 μM, 10 μM, 50 μM, 100 μM. 
Incubate for 1h at 37ºC. 
Proceed to staining.
SECTION: Stain for live NK cells
20. Live (TO-PRO-3-negative) single NK cells (CD56+ CD3-) cells can then be quantified for the amounts of mitochondrial stains relative to negative controls (DMSO and CCCP).