Box 1 |.

3D spheroid invasive area and circularity quantification

CRITICAL It is important to ensure that spheroid invasion dynamics remain largely unaffected when cells are transduced with photoconvertible tag. (The same principles can be applied to confirm no off-target effects from tag in user assay of choice)

Procedure – Timing 3 days (imaging and spheroid invasion), 1 h (imaging analysis)

1. Establish and embed spheroids with and without photoconvertible tag (Steps 16 – 26).

2. Image spheroid on day 0, day 1, day 2 using Compound light microscope at 4X (Step 27).

3. Transfer imaging data and open FIJI software (or other software of your choice).

4. Set up analysis tools to determine object circularity and surface area. Use the ‘draw’ to create an outline of each spheroid (including invading cells).

5. Calculate circularity and surface area for each experimental group and export data to excel to determine standard deviation between spheroid technical replicates.

6. Compare results to determine statistically distinct differences in invasive area or circularity between naïve cells and those transduced with photoconvertible tag.