Bz x700 analyzer

We disaggregated endometrial tissue specimens donated by hysterectomy patients using the method described above. Each sample (1.0 × 10 7 cells) was washed, suspended in FACS Buffer, and blocked with anti-FcR mAb for 15 min. Next, we suspended it in 100 µl FACS Buffer and stained it for 15 min with FITC anti-human CD9 (#312,104 BioLegend), PE/Cyanine7 anti-human CD10 (#312,214, BioLegend), and APC anti-human IL17RB (#FAB 1207 A, R&D Systems, Minneapolis, MN, USA), after which we removed dead cells using propidium iodide. We then sorted the cells into IL17RB-positive and IL17RB-negative groups using the BD FACSMelody Cell Sorter. We seeded the two groups separately in 48-well plates at a density of 4.5 × 10 3 cells per 5 µl Matrigel and cultured them in ExM (250 µl/well) containing either IL17B only (100 ng/ml), SP600125 only (5 µM), or both. We grew these cultures at 37 °C under 5% CO 2 for 7 d, during which we recorded them using time-lapse photography under a fluorescence microscope (BZ-X800, Keyence). We quantified the cultures in terms of organoid count (i.e., organoids ≥ 20 μm in diameter) per unit volume in Z-stacked images using a BZ-X700 microscope and BZ-X analysis software (BZ-X700 Analyzer, Keyence).

To evaluate cell migration ability, several approaches, such as Transwell assays and wound healing assays, can be used. In this study, we used wound healing assays, which have been used in many other studies, to evaluate the effects of TIMP-1 on the colon cancer cell migration (30) (31) (32) . The LoVo, HT29, and HCT116 cells were cultured in DMEM supplemented with 2% FBS in 24-well plates until confluency. The wounds were carefully generated by scratching the confluent cells with the 200-µl pipette tips. The cells were washed with PBS. Next, the cells were cultured in DMEM containing 2% FBS and human recombinant TIMP-1 or the control DMEM containing equivalent amount of BSA prepared in PBS. The images from the wound-healing assay were captured and analyzed using the BZ-X700 and BZ-X700 Analyzer (Keyence) at 0, 24, 48, and 72 h.
The effect of TIMP-1 neutralization on cell migration was evaluated by wound-healing assay. The assay was performed in the presence of human recombinant TIMP-1 and in the presence or absence of human TIMP-1 neutralizing antibody or in control DMEM/PBS.

We treated the cultured organoids with Cell Recovery Solution to remove the Matrigel, agitated them by pipetting, and trypsinized them using TrypLE Express. We counted the disaggregated cells using a hematocytometer grid and then seeded them on a 24-well plate at a density of 3.5 × 10 4 cells per 10 µl Matrigel. To each well, we added 500 µl ExM containing one of three recombinant human cytokines: IL6 (#200-06, PeproTech, Cranbury, NJ, USA), IL8 (#200-08, PeproTech), or IL1β (#200-01B, PeproTech). We maintained the cultures for 10-20 d, exchanging the cytokine-containing medium every 48 h. Finally, we observed them using an all-in-one fluorescence microscope (BZ-X710: Keyence, Osaka, Japan) to check for cytokine-associated differences in organoid-forming efficiency, which we quantified as the number of organoids (i.e., ≥ 20 μm in diameter) per unit volume in Z-stacked images (2.28 × 10 -1 mm 3 ) using BZ-X analysis software (BZ-X700 Analyzer, Keyence).