Operetta cls high content imaging system

Differentiated 3T3-L1 were washed with PBS and incubated with complete medium containing 200 nM of MitoTracker™ Deep Red (Thermo Fisher Scientific) for 30 min. Images were obtained using an Operetta CLS High-Content Imaging System (Perkin Elmer, Waltham, MA, USA) and digitalized using harmony software (Perkin Elmer).

Cells on micropatterned substrates were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min at room temperature and then permeabilized with 0.2% (v/v) Triton X-100 (Sigma-Aldrich) in PBS for 10 min at room temperature. Fixed samples were incubated in blocking buffer (10% (v/v) goat serum (Sigma-Aldrich) and 0.25% (v/v) cold water fish skin gelatin (Sigma-Aldrich) in PBS) for 1 h at room temperature. Substrates were then incubated in primary antibodies diluted in blocking buffer overnight at 4ºC. Primary antibodies used in this study are listed in Supplementary Table 1. After washing thoroughly with PBS, samples were incubated with Alexa Fluor-conjugated secondary antibodies (Alexa Fluor 488, 594 or 647, Invitrogen) and 4,6′-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific) for 1 h at room temperature, protected from light. Micropatterned plates were then washed with PBS before proceeding to imaging. Images were acquired on an Operetta CLS high-content imaging system (10× objective, PerkinElmer) and an A1R confocal microscope (40× objective, Nikon). Confocal images were processed using Icy software (Quantitative Image Analysis Unit, Institut Pasteur, Paris, France).

4T1 cells were cultured in 96-well Cell Carrier microplate (PerkinElmer) (4 × 10³ cells per well) overnight at 37 °C in a humidified 5% CO₂ atmosphere. LET-11 (30 μM) or LET-11-Fe (30 μM) was incubated with 4T1 cells for 4 h. After laser irradiation (808 nm, 0.3 W cm⁻² for 10 min), the cell migration, speed, displacement, and roundness were recorded under digital phase contrast model using an Operetta CLS High-Content Imaging System (Operetta, PerkinElmer, USA), which quipped with a temperature and CO₂ control option set to 37 °C and 5% CO₂. Images were acquired for up to 12 h and recorded every 1 h. All data were processed through Harmony 4.8 Software.

CHOK1 cells stably expressing human GIPR (CHOK1 + GIPR) and CHOK1 cells were cultured in Ham’s F12 (Gibco) supplemented with 10% FBS, 1% penicillin/streptomycin/glutamine (PSG) with or without 5 μg/ml puromycin. Cells were plated at a density of 20,000 cells/well in 96-well plates and cultured for 4 h at 37 °C, 5% CO₂ to allow cell attachment prior to treatment with vehicle (culture media) or 100 nM DA-GIP. After 24 h of treatment, cells were washed three times prior to acclimation to assay buffer (F12 + 0.1% BSA) for 1 h at 37 °C, 5% CO₂. The cells were then placed on ice for another 15 min prior to treatment with a dose titration of Rhodamine GIP (8 nM to 2 µM, Phoenix Pharmaceutical) in ice-cold F12 + 0.1% BSA. Cells were incubated with Rhodamine-GIP on ice for 60 min and then washed three times with cold PBS and fixed with 4% paraformaldehyde. Cells were then washed and stained with Hoechst 33342 (Thermo Fisher) for nuclei detection. Cells were imaged with Operetta CLS high content imaging system (Perkin Elmer) and Rhodamine-GIP fluorescence was quantitated using the Harmony analysis software (Perkin Elmer). Data represented as relative fluorescence unit (RFU) with background fluorescence (calculated from CHOK1 cells) subtracted.

1 × 10⁴ Akata cells were seeded in a 96-well plate in 180 μl RPMI 1640 with 10% FBS and incubated with 20 μl twofold serially diluted CNE2-EBV-GFP virus at 37 °C in a 5% CO₂ humidified atmosphere. After 48 h incubation, the plate was shaken to disperse the cells and let them be evenly distributed in the well. Images were captured and the total GFP positive spots of each well were calculated using the Operetta CLS high content imaging system (PerkinElmer).

Enteroids were fixed with 4% paraformaldehyde at the end of differentiation and recovered from the matrix for whole-mount immunofluorescence staining.⁵² Antigen-retrieval was performed using 10 mM sodium citrate pH 6.0 for 30 min at 80°C followed by pre-incubation (2 h) with 10% donkey serum (Jackson ImmunoResearch) and 0.3% Triton X-100 (Sigma-Aldrich). Enteroids were then incubated overnight at 4°C with goat anti-chromogranin A (1:250; sc-1488, Santa Cruz), goat anti-ghrelin (1:250; sc-10368, Santa Cruz), rabbit anti-GLP-1 (1:100; ab108443, Abcam), rabbit anti-motilin (1:50; in-house developed antibody) or rabbit anti-Notch1 (1:100; D1E11, Cell signaling Technology). After washing, enteroids were stained during 2 h with a secondary antibody CY3 donkey anti-goat (1:800; 715-165-150, Jackson ImmunoResearch) or CY5 donkey anti-rabbit (1:800; 711-175-152, Jackson ImmunoResearch), depending upon the host of the primary antibody. Nuclei were stained with DAPI (2.5 μg/ml, ThermoFisher Scientific). For the negative control, the primary antibody was replaced with normal rabbit serum (Jackson ImmunoResearch). Enteroids were mounted back in a 96- CELLSTAR®-plate (Greiner) with Mowiol® 4-88 (Sigma-Aldrich) and visualized using the Operetta CLS High-Content Imaging system (Perkin Elmer). Counting was performed on at least 3-4 views from ± 10 enteroids per patient.

For cell adhesion and migration assays, tissue culture plates were coated with collagen (5mg/cm², collagen I from rat tail tendon in 0.02N acetic acid) or (5mg/cm², Corning) in PBS overnight at +4°C or 1h at +37°C. Serum was not used in adhesion assays. Cell-cell clustering and plate-and-wash assays were extensively washed with PBS two hours after seeding 10 000 cells per well in 96-well plates. The cells were fixed (10min, 4% PFA) and stained with DAPI and Phalloidin-488 (Fisher Scientific). Images were acquired with the PerkinElmer Operetta CLS High-Content Imaging System and analyzed using custom algorithms in the PerkinElmer Harmony high-content analysis software package (Figure S5A; STAR Methods). Migration was assayed with a Phasefocus LivecyteTM (Phase Focus Limited) on sub confluent 96-well plates. For the real time cell adhesion experiment, cells were detached with 5 mM EDTA and the adhesion of 15 000 cells to fibronectin (5 mg/cm2 in PBS, o/n, +4°C, Sigma-Aldrich) or BSA (1 mg/ml in PBS, o/n) coated wells was measured by xCELLigence RTCA (ACEA Biosciences Inc.).