Ethylene diamine tetraacetic acid (edta)

Isopropanol, ethanol, sucrose, glycerol, Tris, EDTA‐2K, potassium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from Fujifilm Wako Pure Chemical Co.. Meanwhile, 1 M Tris‐HCl (pH 9.0), 0.5 M EDTA (pH 8.0), 10% SDS, TE buffer, TE‐saturated phenol, phenol/chloroform/isoamyl alcohol (25:24:1), and 3 M sodium acetate (pH 5.2) were purchased from Nippon Gene Co., Ltd..

In this study, we modified the extraction method of Sahara et al.[19 (link)]. Spinal cord tissues were homogenized in 19 volumes (w/v) of extraction buffer containing 50 mM Tris-HCl (pH7.5), 5 mM EDTA (Nippon Gene, Tokyo, Japan), 1 mM EGTA (Nacalai tesque), 1% NP-40 (Sigma Aldrich), 0.25% deoxycholic acid sodium salt (Sigma Aldrich), 0.1 M NaCl, 0.5 mM PMSF (Sigma Aldrich), 1 × PhosSTOP (Roche, Basel, Schweiz), and 1 × Complete EDTA(-) (Roche). Homogenates were centrifuged at 250,000g at 4°C for 20 minutes. The supernatants were collected as the tris buffer-soluble fraction (S1), and 10 volumes (tissue weight/volume) of sarkosyl buffer containing 10 mM Tris-HCl (pH7.5), 0.5M NaCl, 1 mM EGTA, 10% sucrose (Wako Pure Chemical), and 1% sarkosyl were added to precipitates followed by sonication. The solutions were incubated at 37°C for 60 minutes, and centrifuged at 250,000g at 4°C for 20 minutes. The supernatants were collected as the sarkosyl-soluble fraction (S2), and 10 volumes (tissue weight/volume) of PBS (Nacalai tesque) were added to precipitates followed by sonication (sarkosyl-insoluble fraction, P2).

Cell suspensions of the small intestine were prepared as previously described^{33 (link)}. After removal of Peyer’s patches, small intestines were incubated on a shaker in PBS containing 2% FBS (complete medium; CM) and 1 mM DTT (Sigma-Aldrich, St. Louis, MO) at 37 °C for 20 minutes and subsequently incubated with 1.3 mM EDTA (Nippon Gene, Tokyo, Japan) in CM at 37 °C for 1 hour. After harvesting the supernatants containing IECs, the rest of the intestinal specimens were digested with 0.3 mg/ml type IV collagenase (Sigma-Aldrich) at 37 °C for 1 hour, homogenized, and filtered. To separate serosal cells and lamina propria cells, the intestinal muscularis covered by the serosa was peeled off before digestion using fine surgical forceps and a dissection microscope^{34 (link)}. The cell suspension was stained with antibodies listed in Supplementary Table 1. Dead cells were removed from the analyses as DAPI (Dojindo Laboratories, Kumamoto, Japan)-positive cells. Cells were analyzed with a FACS CantoII (BD Biosciences, San Jose, CA). Fluorescence-activated cell sorting was performed using a FACS Aria II (BD Biosciences).