Table 6.

Workflow and Timelines of FIS assay validation.

Fis Assay Validation - Workflow & Timelines (Suggested)
Wk nr	Action	Extra comments
> Wk 0	Prepare all media prior to starting the reference organoid cultures.
Wk 0	Thaw 1 cryovial into 3–4 wells (pre-warmed 24-well plate) per reference organoid line.	•Check the seeding density under the microscope. •Add ROCKI (10 μM) to CM +/+.
Wk 1	If budding organoid structures are visible: → Disruption 1–2 full well and seed into 4 fresh wells If organoids are still very small: → Take up all 4 wells, centrifuge and seed into 4 fresh wells	•Switch to regular CM +/+. •Skip organoid clean up step.
Wk 2	If budding organoid structures are visible: → Passage 1–2 full wells of organoids into 4 new wells → Pool organoids from 2 wells and freeze a minimum 2 cryovials WCB If organoids are still very small: → Take up all 4 wells, centrifuge and seed into 4 new wells	•Skip organoid clean up step during passaging.
Wk 3	All colon organoid structures should be budding, proliferating structures (Ch. 7, pic 6): → Passage 1–2 full wells of organoids into 4 new wells → Pool organoids from 2 wells and freeze a minimum 2 cryovials WCB	•Skip organoid clean up step during passaging.
Wk 4	Take up 3–4 full wells: → Follow the organoid disruption & clean up steps. → Seed 8 wells per reference line for FIS assay experiment n=1 in wk 5.
Wk 5	Take up 8 full wells: → Follow the organoid disruption & clean up steps. → Seed 64 wells (in a 96 well plate) per reference line and perform FIS assay experiment n1 → Seed 8 wells (in a 24 well plate) per reference line for FIS assay experiment n=2 in wk 6.	•Check if confocal settings and image analysis are optimal.
Wk 6	Take up 8 full wells: → Follow the organoid disruption & clean up steps. → Seed 64 wells (in a 96 well plate) per reference line and perform FIS assay experiment n2 → Seed 8 wells (in a 24 well plate) per reference line for FIS assay experiment n=3 in wk 7.
Wk 7	Take up 8 full wells: → Follow the organoid disruption & clean up steps. → Seed 64 wells (in a 96 well plate) per reference line and perform FIS assay experiment n3 → Seed 4 wells (in a 24 well plate) per reference line for back up.
Wk 7–8	Data analysis: → Average data of FIS experiments n1, n2 and n3 of all reference lines into n=3 → Compare data to FIS results as published. → If finished, stop organoid cultures.	•FIS data should be comparable to Dekkers et al (2016).