35 mm confocal dishes

SW1088 and SW1783 cell suspensions (3.0 × 104 cells/ml) were plated on 35 mm confocal dishes (Biosharp). Then, bromodeoxyuridine (BrdU) (Roche) with U73122, DMSO, or PBS was added to the medium. After 48 h, cells were fixed with 70% acidic ethanol for 20 min at 4 °C, followed by incubation with primary anti-BrdU antibody (ab6326, Abcam), then Alexa-488 secondary antibody, and finally DAPI. BrdU- and DAPI-positive cells were then counted in ImageJ.
For cell cycle analysis, cells were serum starved for 24 h, and then the medium was replaced with complete growth medium. After 12 or 24 h, cells were collected for DNA staining using propidium iodide (PI), and DNA content was analysed by using a BD Canto II Flow Cytometry System (BD Biosciences) and FlowJo software, v10.6.
For the apoptosis assay, cells were serum starved as described above and then freshly collected and stained for early apoptosis markers using a fluorescein isothiocyanate (FITC) annexin V apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. Annexin V and PI staining were measured using a flow cytometer and analysed using FlowJo v10.6.

MH-S cells were grown in 35-mm confocal dishes (BioSharp, China). After incubating with IL-4 (20 ng/ml) for 6 h and the removal of the culture medium, the cells were washed twice with PBS for 5 min, fixed in 4% paraformaldehyde at room temperature for 15 min, washed again twice with PBS for 5 min, then permeabilized with 0.3% Triton X-100 in PBS for 30 min. After being washed twice with PBS for 5 min, the cells were blocked with 10% goat serum at room temperature for 2 h and incubated with primary antibody for CTFG (1:200) or TGF-β1 (1:200) overnight at 4°C. The next day, after being kept at room temperature for 1 h, the cells were washed three times with PBS and then with an appropriate fluorophore-conjugated secondary antibody for 1 h at room temperature in the dark. After being washed twice with PBS and once with ddH₂O, DAPI (100 nM) was used to stain the cells’ nuclei for 2 min in the dark. Finally, after being washed once with ddH₂O, the cells were kept in a mounting medium before images were captured [21 (link)] using a laser scanning confocal microscope (Carl Zeiss LSM700, Germany).