Ethylene diamine tetraacetic acid (edta)

The cells were harvested following brief exposure to 0.25% trypsin/1 mM EDTA (Nacalai Tesque, Inc.). The cells were washed with 0.1% BSA/PBS and treated with primary mAbs for 30 min at 4 °C. Thereafter, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) or Oregon green anti-rat IgG (1:2000; Thermo Fisher Scientific Inc.). Fluorescence data were collected using SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).

CHO/mEGFR, CHO-K1, NMuMG, and Lewis lung carcinoma were harvested after a brief exposure to 1 mM ethylenediaminetetraacetic acid (EDTA, Nacalai Tesque Inc.). The cells were treated with EMab-300 or blocking buffer (control) (0.1% BSA in PBS) for 30 min at 4 °C, followed by treatment with Alexa-Fluor-488-conjugated anti-rat IgG. The data were analyzed using the SA3800 Cell Analyzer and SA3800 software ver. 2.05 (Sony Corp., Tokyo, Japan).

iPS cells were removed from dishes at 80% confluence using CTK solution and washed DPBS (−). The removed cells were treated with 0.5× TrypLE Select solution. 0.5× TrypLE Select solution was prepared with 50% TrypLE Select, no phenol red (Thermo Fisher Scientific) and 0.25 mM EDTA (0.5 mol/L EDTA Solution (pH 8.0), Nacalai Tesque Inc.) in DPBS (−) for single-cell culture. The cells were collected in the medium with 10 μM Y-27632 (Fujifilm Wako Pure Chemical Corporation) and centrifuged. The supernatant was removed before use.
Feeder cells were seeded with 1.25 × 10⁴ cells/well in a 96-well plate and cultured for 1 day in the medium including bFGF and Y-27632, and iPS cells were additionally seeded with 5000 cells/well and cultured for 1 day. The culture media was substituted by those supplemented with DMSO (CultureSure DMSO, Fujifilm Wako Pure Chemical Corporation) or ZIL. The cells were cultured until the control sample (which does not contain the solvents) became 80% confluent. The cell viability was confirmed by using Cell Count Reagent SF (Nacalai Tesque Inc.). The culture media was substituted by the media supplemented with 10% of Cell Count Reagent SF and incubated for 1 h. After incubation, absorption at 450 nm was measured and translated to the cell viability with a standard curve.

FACS analysis and cell sorting were performed as described previously [48] (link). In brief, the small intestine was cut longitudinally and washed in cold PBS. Then, a 2-cm piece of intestine was cut and incubated in 2% (vol/vol) fetal calf serum in RPMI 1640 (Wako) containing 0.5 mM EDTA (Nacalai Tesque) for 15 minutes at 37°C. Cells were filtered with a 70-μm cell strainer (Becton Dickinson) and blocked with Fc Block (2.4G2; Becton Dickinson) for 5 minutes at room temperature, followed by staining antibodies and reagents for 30 minutes at 4°C. Dead cells were excluded by propidium iodide (PI) staining. Epithelial cell fraction was defined by PI⁻CD45⁻TER119⁻EpCAM⁺ gating and biotinylated HKH-189 was used for Cld4 staining. Samples were analyzed by FACSCanto (Becton Dickinson) or sorted by FACSAria (Becton Dickinson).

Cultured cells were washed with PBS, collected using a cell scraper into a triglyceride assay buffer [5% Triton X-100 (Nacalai Tesque), 1 mM EDTA (Nacalai Tesque), and 25 mM Tris-HCl pH 7.5], and lysed by sonication. The triglyceride contents in the cell lysates and supernatants and the apo B contents in the supernatants were detected using a Triglyceride Assay Kit (BioAssay Systems, Hayward, CA, USA) and a Total Human Apo B ELISA Assay Kit (ALerCHEK Inc., Portland, ME, USA), respectively, according to the manufacturer's instructions. The absorbance signals were measured with a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Hec1 cells were harvested using 0.25% trypsin/1 mM EDTA (Nacalai Tesque) solution and evaluated for aldehyde dehydrogenase (ALDH) activity using the ALDEFLUOR assay kit per the manufacturer's instructions (STEMCELL Technologies, Vancouver, Canada). Flow cytometry was conducted using the FACS Caliber or FACSVerse (BD Biosciences, San Jose, CA).

We obtained AM from women undergoing cesarean section. We collected tissue from AM for our clinical purposes. We sufficiently explained the proposed use to the women and obtained their informed consent. We washed the membranes under aseptic conditions with phosphate-buffered saline (PBS) containing 5 ml of 0.5% levofloxacin and stored them at −80°C in Dulbecco’s modified Eagle’s medium (GIBCO/Invitrogen, Carlsbad, CA, USA) and glycerol (1:1, v:v; Wako Pure Chemical Industries, Ltd., Osaka, Japan). AM was thawed immediately before use for the oral epithelial cell culture and washed three times with PBS. In the oral epithelial cell cultures, membranes were deprived of their amniotic epithelial cells by incubating with 0.02% EDTA (Nacalai Tesque, Inc., Kyoto, Japan) at 37°C for 2 h to loosen cellular adhesion, followed by gentle scraping with a cell scraper (Nalge Nunc International, Naperville, IL, USA) to remove the amniotic epithelial cells.