TITLE: Assessment of oxidative phosphorylation and glycolysis in NK cells (Seahorse assays)
AUTHORS: Philippa R Kennedy

[DESCRIPTION]
The rate of glycolysis (as measured by acidification of the extracellular space) and the rate of oxidative phosphorylation (as calculated from the consumption of oxygen) are measured in cells before and after the addition of drugs that test the resting and maximal capacity of these cells to respond.

Agilent offers a Mito Stress test kit (cat. 103015-100) to analyze both extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in a combined assay that is the most commonly used in the literature. This assay is conducted in the presence of glucose. The glycolytic rate assay (cat. 103344-100) and ATP rate assays (cat. 103592-100) determine basal ECAR in the media that initially does not contain glucose and thus gives a more precise measure of glycolysis.

[STEPS]
SECTION: Day 1
6. NK cells are resuspended in Seahorse XF Assay Medium and added to a 96 well or 24 well flat bottom plate that has been coated with 0.01% poly-L-Lysine solution (Millipore Sigma) according to the manufacturer's instructions.
SECTION: Day 1
9. The extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) are measured (pmoles/min) in real time in an XFe96 analyzer after injection of drugs in the specified order. 

e.g. 
Mito Stress Test kit (10 mM glucose, 1 μM oligomycin then 1 μM FCCP plus 1mM sodium pyruvate then 0.5 μM rotenone/antimycin A; Cat. No. 103015-100)
Glycolytic rate assay kit (0.5μM rotenone/antimycin A then 50mM 2-DG; Cat. No. 103344-100) 
ATP Rate Assay kit (1μM oligomycin then 0.5μM rotenone/antimycin A; Cat. No. 103592-100; all Agilent Technologies)
SECTION: Day 1
10. The spare respiratory capacity (SRC) is calculated from the change from basal oxygen consumption, after addition of glucose, to maximal oxygen consumption, after addition of FCCP. 
SECTION: Day 0
1. Day before assay: Hydrate seahorse sensor cartridge

Add 200 μL of sterile water to each well of the Seahorse utility plate. 

SECTION: Day 1
3. Day of assay:

Replace water in utility plate with 200 μL Seahorse calibrant per well and place back into the non-CO2 incubator
SECTION: Day 1
5. Wash NK cells twice, once with PBS and once with Seahorse XF Assay medium (Agilent Technologies)
SECTION: Day 1
7. Spin down cells to adhere to the plate. Spin plate at 40 g (500 RPM) for 4 minutes. Rotate plate 180° and then spin cells at 80 g (700 RPM) for 4 minutes without brake. 



SECTION: Day 1
7.1. Check for even plating under microscope. 
SECTION: Day 1
4. Make sure that the Seahorse XF Assay medium (Agilent Technologies) is at pH 7.4
SECTION: Day 1
8. Prepare drugs and add 25 μL into ports for a 96 well seahorse assay or 75 μL into ports for a 24 well seahorse assay
SECTION: Day 1
7.2. Put cell plate in the non-CO2 incubator while preparing drugs.
SECTION: Day 1
6.1. Option 1: 500,000 cells/well are added to a 96 well plate.
SECTION: Day 1
6.2. Option 2: 1 million cells/well are added to a 24 well plate.
SECTION: Day 0
2. Add Seahorse Calibrant to a 50 mL conical tube. Place both the cartridge and aliquoted calibrant at 37°C in a non-CO2 incubator overnight.