A Two-Population Experiment

In this tutorial, you will analyze a co-culture experiment: MCF-7 cancer cells (targets) and primary NK cells (effectors). We will focus on the targets to detect lysis events.

Step 1: Get the demo data

We provide a demo experiment in Zenodo (ADCC experiment).

  1. Open your terminal and run:

    $ python -m celldetective

  2. In the menu bar, go to File > Open Demo > Cytotoxicity Assay Demo.

  3. The project will download and load automatically.

Step 2: Segment Targets

We will process the first position of the first well.

  1. In the top control panel, select Well W1 and Position 100.

  2. Expand the PROCESS TARGETS block.

  3. Check the Segment box.

  4. In the Model zoo, select mcf7_nuc_stardist_transfer.

  5. Click Submit to run segmentation.

    Tip

    Click the button to visualize the segmentation results in Napari.

Step 3: Track Targets

  1. Check the Track box.

  2. Click the button to configure tracking.

  3. Set parameters:

    • Minimum tracklength: 10

    • Check Remove tracks that do not start at the beginning.

    • Check Interpolate missed detections.

    • Check Sustain last position.

    • Uncheck other post-processing options.

  4. Click Save.

  5. Ensure Segment is unchecked and Click Submit.

Step 4: Measure Targets & Detect Events

We will detect cell death (lysis) using the uptake of Propidium Iodide (PI).

  1. Check the Measure box.

  2. Check the Detect Events box.

  3. In the Signal models list, select lysis_PI_area.

  4. Click Submit.

Step 5: Process Effectors

We will segment and measure the primary NK cells (Effectors) without tracking them, as the time resolution is low.

  1. Expand the PROCESS EFFECTORS block.

  2. Check the Segment box.

  3. In the Model zoo, select primNK_cfse.

  4. Ensure Track is unchecked.

  5. Check the Measure box.

  6. Click Submit.

Step 6: Analyze Interactions

Compute the proximity between Targets and Effectors.

  1. Expand the INTERACTIONS block.

  2. Check the Neighborhoods box.

  3. Click the button next to ISOTROPIC DISTANCE THRESHOLD.

  4. Set Distance to 30 pixels.

  5. Click Add.

  6. Click Submit.

Step 7: Analyze Time-Series

Visualize the single-cell signals and the detected lysis events.

  1. Click the button in the Detect Events section (Event Annotator settings).

  2. Configure the RGB representation if needed and click Save.

  3. Click the button (Event Annotator) to open the viewer.

  4. You can inspect the traces and see the detected lysis times.

Note

Since we computed neighborhoods, you can also visualize the number of effectors in contact/proximity with each target cell. Look for the inclusive_count_neighborhood_(targets-effectors)_circle_30_px signal in the list.

signal_annotator

Visualize single-cell signals (e.g. intensity, neighbors) with the signal annotator.

Step 8: Explore Results

  1. Survival Analysis:

    • Go to the Analyze tab.

    • Click Plot survival.

    • Select Population: targets.

    • Time of Interest: t_lysis.

    • Time of Reference: 0 (beginning of experiment).

    • Click Submit to view the Kaplan-Meier survival curve.

  2. Signal Synchronization:

    • Click Plot signals.

    • Select Population: targets.

    • Class: class_lysis.

    • Time: t_lysis (to align signals at the moment of death).

    • Click Submit to view the average lysis signature.

Next Steps