Confocal microscopy of intracellular components within NK cells
Philippa R Kennedy
Abstract
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.
Steps
Coat a plate with fibronectin
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
When ready to continue, quickly rinse wells three times with PBS to remove excess fibronectin.
Allow NK cells to adhere to coverglass
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.
Stain the cells
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.
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.
Permeabilize
- 400 μL 0.1% Triton-X/PBS for 5 min at room temperature.
- Gently rinse twice with PBS
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).
Imaging
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.
Analysis
Denoise
e.g. Nikon Elements Fourier transform.
Model
e.g. Imaris Spot detection can model mitochondria. Membrane and nuclear dyes help model the whole cell structure around these mitochondria.
