Heracell 240i

The human colorectal adenocarcinoma cell line Caco-2 (ATCC; HTB-37; DSMZ; ATCC169, ECACC09042001, ECACC; 86010202, Beijing, China) was used in this study. The culture fluid was aspirated from the cell culture flask and allowed to stand in a CO₂ cell incubator (HERAcell 240i; Thermo Fisher Scientific; Waltham, MA, USA); remaining medium in the bottle was aspirated, and phosphate-buffered saline (PBS; Solarbio Technology Co., Ltd., Beijing, China) was used to wash the cell surface before adding 2 mL 0.25% trypsin-EDTA (Lot no: 2186970; Gibco, Grand Island, NY, USA) to digest the cells for 2–4 min after aspiration. The cell status was observed by inverted microscope (Caikang Optical; Shanghai, China) before adding MEM/EBSS-10 medium (Lot No: AG29584972; Cytiva, Marlborough, MA, USA) and fetal bovine serum (FBS, Lot No: 1907301; v/v:90/10) to stop the intestinal digestion. Thereafter, the cells were digested in a 100 mm cell culture dish until the cells adhered to the wall and reached a confluence of approximately 80%. This process was three times repeated, and the passaging ratio was 1:3.

Cytotoxicity assay was performed using CellTox™ Green Cytotoxicity assay kit (Promega, Mannheim, Germany). Briefly, cells were cultured in 96-well plates at the density of 25 × 10³ cells/well in DMEM medium containing 10% fetal calf serum (Biochrom AG, Berlin, Germany) and antibiotics (40 U/mL penicillin and 40 μg/mL streptomycin, both from PAA Laboratories, Linz, Austria). Cells were pre-treated with different concentrations of AM404 (0.1–10 μM) or DMSO 0.1% for 30 min. Thereafter, cells were incubated with or without LPS for the next 24 h. Ethanol (10% end conc., Sigma-Aldrich, Taufkirchen, Germany) was used as positive control to induce the cell death. After incubation (10% CO₂ at 37 °C - Heracell 240i, Thermo Scientific), 100 μl of CellTox™ Green reagent were added in each well. The plate was mixed for 1 min and incubated for 15 min at room temperature, and the fluorescence was measured at 490 nm_Ex/530 nm_Em using a Modulus™ II Microplate Multimode Reader (Turner BioSystems, USA).
The principle of the assay is to evaluate the alterations in the membrane integrity, using the cyanine dye. The dye binds in the dead-cell DNA and enhanced the fluorescent property, which is excluded from viable cells. The fluorescence intensity values obtained were normalized and presented as the percentage of untreated controls.

To obtain pre-implantation embryos, female mice were superovulated by intraperitoneal injection of 5 or 7.5 international units (IU) of pregnant mare’s serum gonadotropin (Intervet, Intergonan) followed by 5 or 7.5 IU of human chorionic gonadotropin (hCG; Intervet, Ovogest 1500) 48 hours later. Hormone injection dosage was batch dependent and determined by LAR services. Superovulated females were mated with male mice directly after hCG injection. Embryos were flushed from dissected oviducts and uteri of female mice after super-ovulation and mating with male mice. Embryos were flushed from dissected oviducts and uteri at 48, 72, 78, 84, 96 and 108 hours post-hCG using KSOMaa with Hepes (Zenith Biotech, ZEHP-060). After flushing, embryos were washed in KSOMaa with Hepes, transferred to 10μL drops of KSOMaa (Zenith Biotech, ZEHP-050) covered with mineral oil (Sigma, M8410) on either a tissue culture dish (Falcon, 353001) or petri dish (Falcon, 351008) and then cultured at 37^°C in a CO₂ incubator (Thermo Scientific, Heracell 240i) with 5% CO₂.