Zen analysis software

Data analysis was performed using the Zen analysis software (Carl Zeiss AG, Germany) as well as Fiji (ImageJ) [39 ]. Cell tracking was performed using the TrackMate plugin (v.2.8.2) for Fiji [40 (link)] and three-dimensional visualisations of the data were generated using the 3D Viewer (v.) plugin for Fiji [41 (link)]. The TrackMate [42 (link)] plugin for Fiji is a single particle tracking tool that identifies spots, i.e. cells, in every frame and the trajectories of cells are reconstructed by assigning an identity over these frames in the shape of a track. The positional data and numerical features generated using TrackMate were further processed and displayed using Matlab (R2014a, The Mathworks Inc., USA). Violin plots were generated using a script obtained via Matlab">https://github.com/bastibe/Violinplot-Matlab. The Mann-Whitney U procedure for statistical testing was run in Matlab. Multiview reconstruction was performed using the MultiView Reconstruction plugin in Fiji [43 , 44 (link)]. Object segmentation and volume calculation of the segments was done in Imaris (8.4.2, Bitplane AG, Switzerland).

Media was aspirated away from Matrigel domes. Matrigel domes were washed with 500 μL room temperature PBS. Wells were fixed with 4% paraformaldehyde in PBS for 30 mins at room temperature. Wells were rinsed 3 × 10 min with 500 μL 100 mM glycine in Tris pH 7.4. Cells were permeabilized with 500 μL 0.5% Triton X-100 in PBS at room temperature for 5 min. Wells were washed in IF wash buffer (0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20) 3 × 10 min. Wells were incubated with 500 μL of IF wash buffer with 1% BSA to block for 1 h at room temperature. Block buffer was aspirated and wells were incubated with primary antibody (mouse anti-αSMA, AbCam; mouse anti-Maspin, BD-Pharmingen; Rabbit anti-PDX1, Cell Signaling Technology; mouse anti-CK19, AbCam) 1:200 in 500 μL block buffer for 1 h at room temperature. Wells were washed 3 × 20 min in IF wash buffer. Wells were incubated with secondary antibody (Anti-Mouse Alexafluor Texas Red, ThermoFisher or Anti-Rabbit Alexaflour 488) 1:500 in 500 μL block buffer for 1 h at room temperature. Wells were washed 3 × 20 min. Wash was removed thoroughly and 150 μL of SlowFade anti-fade mountant with Dapi (ThermoFisher). Organoids were observed and micrographed on a Zeiss inverted microscope with fluorescence and Zen analysis software (Zeiss).

Cells were fixed at RT in 3.8% (vol/vol) formaldehyde in PBS and permeabilized in PBS containing 0.1% (vol/vol) saponin (Sigma-Aldrich) or 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich). Coverslips were labeled with primary and secondary antibodies as previously described (Connell et al., 2009 (link)). In certain cases, soluble cytosolic proteins were removed to allow better imaging of membrane-associated protein, by prefixation treatment with a saponin-based cytosol extraction buffer, as previously described (Edwards et al., 2009 (link)). Slides were analyzed with an LSM880 confocal microscope (100× NA 1.40 oil immersion objective, 37°C), LSM710 confocal microscope (100× NA 1.40 oil immersion objective, 37°C), LSM780 confocal microscope (63× NA 1.40 oil immersion objective, 37°C), or AxioImager Z2 Motorized Upright Microscope (63× NA 1.40 oil immersion objective, RT, Axiocam 506; ZEISS), all with ZEN analysis software (ZEISS). Airyscan imaging was performed using an LSM880 microscope fitted with an Airyscan module, using Plan-Apochromat 63×/1.4-NA Oil differential interference contrast (DIC) M27 objective at RT. Images were subsequently processed using ImageJ, Adobe Photoshop, Adobe Illustrator, and ZEN analysis software. 3D image preparation was performed using Imaris (Bitplane). Colocalization of proteins was quantified using Volocity (PerkinElmer).

Three-dimensional cell culture was performed on 48-well plates coated with Matrigel (356230, Life Sciences, Amsterdam, Netherlands). Wells received 100 µL of Matrigel and plates were left for 30 min at 37 °C for Matrigel gelation. Suspensions of sorted cells were centrifuged at 300 G during 5 min (4 °C) and the cell pellets were resuspended in Epicult medium (Epicult Basal medium, 05602, Stem cell technologies) supplemented with Epicult B supplement (05602, Stem cell technologies), L-Glutamine (2 mM final concentration), hydrocortisone (0.48 µg/mL final concentration) and 2% of Matrigel. Cells were plated at a concentration of 100.000 cells/well and cultured in Epicult medium for 7 days. Cells were observed at 10 × magnification using phase contrast microscopy (Zeiss Axio; Zeiss France, Fougères, France) and images were digitalized. Mammosphere mean diameters were determined from at least 12 frames per conditions using the Zen analysis software (Zeiss, France).