Adhesion assay

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.

Add 200 μl of ice cold 2% paraformaldehyde to individual control samples and store on ice.

Obtain fluorescent effector and target cells and prepare them in fresh pre-warmed media:

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.

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. ⁶live cells/ml if effector cell numbers are not limiting.

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. ⁶live cells/ml if effector cell numbers are not limiting. Prepare drugs e.g. Rituximab at 2X.

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.

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.

Add 100 μl of effector cells. Mix gently, but consistently - e.g. two flicks/tube

Spin tubes at 20 × g for 1 min.

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.

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!

Immediately fix by adding 200 μl of ice-cold 2% paraformaldehyde.

Store samples at 4°C protected from light until analysis.

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.

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.