TITLE: Confocal microscopy of intracellular components within NK cells
AUTHORS: Philippa R Kennedy

[DESCRIPTION]
NK cells are small cells and their organelles e.g. mitochondria are consequently harder to visualize than those in larger cells. We have found that imaging NK cells that are stretched out across fibronectin-coated coverglass spreads out their organelles across a wider area and makes it easier to resolve differences e.g. their mitochondrial surface area and volume. 

There are many possible options for chamber slides, fixatives, permeabilization agents and stains. The protocol here gives specific instructions for a particular nuclear and mitochondrial staining, but the principle can be applied to many different types of stain. 

[GUIDELINES]
Selection of staining reagents
This protocol can be applied for the visualization of various intracellular components. When staining with dyes, make sure the dyes are fixable for microscopy e.g. Mitotracker Deep Red probe, but not MitoTracker Green. Make sure they fluoresce in separate channels e.g. MitoTracker Deep Red FM and Wheatgerm Agglutinin AlexaFluor 647 fluoresce in the same channel on the Nikon A1Rsi microscope described, so should not be used together.

Optimal cell density
Cell density per well depends on the size of the cell when it is spread out on the glass, how many you tend to knock off with your wash steps and what power you are imaging at. You generally want a higher density if imaging at 60x and a lower density if imaging at 10x. If adding directly to fibronectin coated surfaces 200,000-400,000 pNK/cm2 should be about right. If staining in the well, consider doubling the number when plating initially. For experiments where NK cells are cultured at 300,000 cells/200 μL in 96 well U-bottom plate format, we find transferring 2 wells from the U-bottom into a single chamber slide well one day before the end of culture works well.

[STEPS]
SECTION: Coat a plate with fibronectin
1. Coat chambered coverglass (µ-Slide 8 well polymer bottom 1.5, Ibidi, Cat. 80826) with fibronectin (10μg/mL in PBS, Sigma-Aldrich, Cat. F1141) to allow NK cells to stretch out across the coverglass.

Coating options:
30 min at 37°C
45 min at room temperature
overnight at 4°C
SECTION: Allow NK cells to adhere to coverglass
3. Add NK cells onto the coverglass and allow them to adhere. 

A minimum of 20 min at 37°C is required. 
Overnight culture at 37°C is often the simplest option. 
See guidelines for NK cell density e.g. 750,000 cells/well.
SECTION: Coat a plate with fibronectin
2. When ready to continue, quickly rinse wells three times with PBS to remove excess fibronectin.
SECTION: Stain the cells
4. MitoTracker Deep Red FM (Thermo Fisher Scientific, Cat. M22426)
The stock is resuspended in DMSO (Cat. BP231-100, Fisher Bioreagents) and diluted to 200 nM in RPMI (Gibco Cat. No. 2240-089).
Add 400 μL of this solution into each well
Stain for 30 min at 37°C
Gently rinse once with RPMI.
SECTION: Stain the cells
5. Fix the cells
Add 400 μL 4% paraformaldehyde/RPMI for 15 min at 37°C (do not prepare this solution in advance).
Gently rinse once with PBS.
SECTION: Stain the cells
6. Permeabilize
400 μL 0.1% Triton-X/PBS for 5 min at room temperature.
Gently rinse twice with PBS
SECTION: Stain the cells
7. Counterstain
Add 400 μL DAPI (2 drops/mL PBS)
Incubate for 20 min at room temperature. 
Proceed to imaging (no need to wash DAPI or phalloidin stains). 
SECTION: Imaging
8. If no azide is included in the media, proceed to imaging soon after staining is complete (store at 4 °C until required). 
Chamber slides can be imaged with water immersion objectives. 
Within our University Imaging Center, Jackson Hall, there is a Nikon A1Rsi HD confocal microscope with 60x 1.27 NA water immersion objective suitable for this kind of microscopy. 
DAPI is excited by the 405 nm laser and MitoTracker Deep Red FM is excited by the 640 nm laser. 
There are 4 PMT detectors, of which two are highly sensitive GaAsP detectors and a transmitted light detector. 
SECTION: Analysis
9. Denoise
e.g. Nikon Elements Fourier transform.
SECTION: Analysis
10. Model
e.g. Imaris Spot detection can model mitochondria. Membrane and nuclear dyes help model the whole cell structure around these mitochondria.