TITLE: Adhesion assay
AUTHORS: Philippa R Kennedy, Quinlan Kile

[DESCRIPTION]
Adapted from Natural Killer Cell Protocols 2009 p89-96

Enriched effector cells and tumor targets are labelled with different fluorescence. They are allowed to interact over a time-course and samples are taken and fixed at regular intervals. These samples are assessed by flow cytometry for the proportion of firmly adhered NK-tumor conjugates (double positive fluorescent events) at each timepoint.

[BEFORE_START]
Duplicates of each condition are necessary to check the reproducibility of results. Timing is key to reproducibility, so have the bench setup with ice-cold PFA, a vortex, timer and waterbath, all in close proximity.

Calculate the required number of cells for this assay ahead of time
e.g.
8 tubes per target cell per donor (4 timepoints in duplicate): 0, 10min, 20min, 30min. 
3 target cells: K562, Raji + Rituximab, Raji alone
2 culture conditions: 20% oxygen v. 1% oxygen
= 48 tubes per NK cell donor
x 100,000 NK cells/tube = 4.8 million NK cells/donor

[GUIDELINES]
This method does not involve a live/dead stain therefore NK cells and tumor targets must have good viability (>90%) at the start of the assay for results to be meaningful. 

If a drug is being used to increase adherence e.g. Rituximab, consider including the same cell line without the drug to evaluate the contribution of the drug.

We have tested this assay using K562 as a readout of natural cytotoxicity and Raji with Rituximab (Genentech) as a readout of antibody dependent cellular cytotoxicity.

Effector:target ratios will impact the frequency of adherence. A 1:2 ratio used here gives NK cells a high chance of encountering a tumor cell. The experimental readout is the proportion of NK cells in conjugates. Increase the proportion of tumor cells to maximize the likelihood of NK cells encountering a tumor cell (and therefore the limiting factor is its ability to adhere to a target cell). 

Effector cells will be able to adhere to different target types at different rates. When working with a new target cell or effector, optimize the effector:target cell ratio and timing for maximum sensitivity to experimental perturbation.



[STEPS]
SECTION: Set aside cells for flow cytometer setup
1. The following controls will be required to setup the gates on a flow cytometer:
1. Unlabeled cells for an unstained control
2. Single color control (effector) e.g. CellTrace Violet labeled NK cells.
3. Single color control (target) e.g. K562-Nuclight Red.
SECTION: Prepare the cells
3. Obtain fluorescent effector and target cells and prepare them in fresh pre-warmed media:


SECTION: Set aside cells for flow cytometry setup
None. Resuspend K562 in assay medium at 2X million/mL e.g. 2 × 106 live cells/ml if NK cell numbers aren’t limiting. 
SECTION: Set aside cells for flow cytometry setup
None. Resuspend Raji in assay medium at 4X million/mL e.g. 4 × 106 live cells/ml if NK cell numbers aren’t limiting. Prepare Rituximab at 20ug/ml (1/5 dilution of original stock)
SECTION: Set aside cells for flow cytometry setup
None. Controls:
SECTION: Set aside cells for flow cytometry setup
None. Dispense 200 μl of each labeled cell type into a FACs tube to be used for setting up the flow cytometry parameters i.e. NK cells alone, target cells alone controls. 
SECTION: Set aside cells for flow cytometry setup
None. Add 200 μl of ice cold 2% paraformaldehyde to the unlabeled and labeled control samples and store on ice.
SECTION: Set aside cells for flow cytometry setup
None. In duplicate - for each desired time point (0, 10min, 20min and 30min) aliquot 100 μl of K562 cells into FACs tubes; 50ul of Raji into flow tubes + 50ul 20ug/ml Rituximab (add 50uL of R10 to Raji without Rituximab tubes)
SECTION: Set aside cells for flow cytometry setup
None. For the next step: work in batches with small numbers of tubes so that targets+NK cells aren’t sitting around together for too long. Store tubes in the incubator until needed.
SECTION: Set aside cells for flow cytometry setup
None. Add 100 μl of NK cells. Gently mix (be consistent - 2 flicks)
SECTION: Set aside cells for flow cytometry setup
None. Incubate at 37°C in a water bath for desired time points (put them all in together then take them out one at a time at the appropriate timepoint e.g. after 10min; don’t use an incubator here).
SECTION: Set aside cells for flow cytometry setup
None. At the end of each time point, vortex sample at high speed for 3 s (This step can introduce a lot of variation. Don’t vortex too long. It’s the reason you have to do everything in duplicate to start off with!).
SECTION: Set aside cells for flow cytometry setup
None. Immediately fix by adding 200 μl of ice-cold 2% paraformaldehyde.
SECTION: Set aside cells for flow cytometry setup
None. Store samples at 4°C protected from light until analysis.
SECTION: Set aside cells for flow cytometry setup
None. Analyze by flow cytometry as soon as possible (we’ve checked cells stored in the fridge for 5 days and this gave the same result, so you can store them if necessary). Use untreated NK and target cells to set up the forward and side scatter parameters and set an acquisition gate to collect at least 20,000 live cell events. Be sure to use a wide forward scatter gate to capture conjugates, which may have higher forward scatter (see Fig. 7.1 in original protocol). Use the untreated and labeled cells to set up the sensitivity and compensation for the fluorescent channels.
SECTION: Set aside cells for flow cytometry setup
None. Set up the analysis leaving a generous margin for single color events when setting the quadrants. Calculate the % of conjugated NK cells = 2-color events/(2-color events + NK+ only events) × 100%. If desired, the background value can be subtracted from each experimental point which for some cell combinations can be a significant value.
SECTION: Prepare the cells
3.1. Resuspend effector cells at 1 × 106 live cells/ml. If this is not possible, resuspend at Xmillion/mL where X is the concentration of the least abundant condition. 
SECTION: Prepare the cells
3.2. Resuspend tumor cells that do not require drugs e.g. K562, in assay medium at 2X 
e.g. 2 × 106 live cells/ml if effector cell numbers are not limiting. 
SECTION: Prepare the cells
3.3. Resuspend tumor targets that require drugs e.g. Raji, in assay medium at 4X 
e.g. 4 × 106 live cells/ml if effector cell numbers are not limiting. Prepare drugs e.g. Rituximab at 2X.
SECTION: Set aside cells for flow cytometer setup
2. Add 200 μl of ice cold 2% paraformaldehyde to individual control samples and store on ice.
SECTION: Prepare the cells
4. In duplicate - for each desired time point (e.g. 0, 10 min, 20 min and 30 min) aliquot 100 μl of target cells or target cells and drugs (50 µL of each) into flow cytometry tubes.
SECTION: Add effectors to target cells
5. Guidance: For this section, work in batches with small numbers of tubes so that targets and effector cells are not together in the same tube for uncontrolled periods of time. Keeps cells warm, by storing tubes in the incubator until needed.
SECTION: Add effectors to target cells
9. At the end of each time point, vortex sample at high speed for 3 s. 

Guidance: This step can introduce a lot of variation. Have a stopwatch in front of you for precise timing. It is the reason you have to do everything in duplicate to start off with!
SECTION: Run on a flow cytometer
12. Run samples and controls on a flow cytometer. 

Guidance: 
Use single color effector and target cells to set up the forward and side scatter parameters and set an acquisition gate to collect at least 20,000 cell events (not debris). 
Be sure to use a wide forward scatter gate to capture conjugates, which may have higher forward scatter (see an example in the description image). 
Use untreated labeled cells to set up the sensitivity of detectors and compensation for the fluorescent channels.
We have checked cells stored in the fridge for 5 days and this gave the same result as freshly fixed cells, so you can store them if necessary. 
SECTION: Run on a flow cytometer
13. Analyze for the proportion of effector cells in conjugates.

Set up the analysis leaving a generous margin for single color events when setting the quadrants. 
Calculate the proportion NK cells in conjugates = 2-color events/(2-color events + effector only events) × 100%. 
If desired, the background value (proportion of effectors in conjugates at time zero) can be subtracted from each experimental point. For some effector/target cell types, this can be a significant value.
SECTION: Add effectors to target cells
6. Add 100 μl of effector cells. Mix gently, but consistently - e.g. two flicks/tube
SECTION: Add effectors to target cells
8. Incubate at 37°C in a water bath for desired time points. 

Guidance: Put them all in together then take them out one at a time at the appropriate timepoint e.g. after 10 min. Do not use an incubator here. For the 'time zero' control, there is no incubation - proceed immediately to step 9.
SECTION: Add effectors to target cells
10. Immediately fix by adding 200 μl of ice-cold 2% paraformaldehyde.
SECTION: Add effectors to target cells
11. Store samples at 4°C protected from light until analysis.
SECTION: Add effectors to target cells
7. Spin tubes at 20 × g for 1 min.