Pe anti mouse ly 6a e sca 1

BMMCs were plated to the slide and were fixed by heating with 10% (w/v) neutral buffered formalin solution. Cells were reacted with mouse anti-mast cell tryptase antibody (AB2378, Abcam, Cambridge, UK). Cells were then reacted with FITC-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and 1µg/mL DAPI (4′,6-diamidino-2-phenylindole dihydrochloride). Paraffin sections were reacted with respective antibodies and analyzed as described previously [53 (link)]. Sections were subjected to immunohistochemistry using antibodies of Alexa Fluor 488^®-anti-mouse Il-10 (#505013), Brilliant Violet 421™-anti-mouse TGF-β1 (#141407), Pacific Blue™-anti-mouse IL-2 (#503820), Alexa Fluor^® 488-anti-GATA3 (#653808), PE-anti-mouse Ly-6A/E (Sca-1) (#108108), PE-anti-mouse IL-33Rα (ST2) (#146608) and PE-anti-mouse/human IL-5 (#504304) (BioLegend, San Diego, CA, USA), FITC-anti-mouse/human CD45R (#11-0452-82) (Thermo Fisher Scientific, Waltham, MA, USA), PE-anti-mouse CD25 (#130-120-766) (Miltenyi Biotech, Bergisch Gladbach, Germany). All antibodies were used at a 1:500 dilution.

The cell surface markers of the BMMSCs and Mφs were analyzed by flow cytometry. Briefly, the cells were trypsinized and washed with PBS. To block Fc receptors, the cells were incubated with 2% anti-mouse CD16/32 (BioLegend, San Diego, CA, USA) on ice for 10 min. Then, the cells were washed twice with PBS and incubated with specific antibodies for 30 min at 4 °C in the dark. Excess antibody was removed by washing the cells with PBS. Untreated cells were used as blank controls. The samples were then analyzed with a Beckman Coulter Epics XL cytometer (Beckman Coulter, Fullerton, CA, USA). For the characterization of BMMSCs, the following antibodies were used: PE anti-mouse Ly-6A/E (Sca-1), PE anti-mouse CD90.2, PE-anti-mouse CD73, PE anti-mouse CD105, PE anti-mouse CD34 and FITC anti-mouse CD45 (all from BioLegend, San Diego, CA, USA). FITC anti-mouse CD86 and PE anti-mouse CD206 (both from BioLegend, San Diego, CA, USA) were used for the identification of Mφs. In this experiment, pulse width measurements were used to eliminate the possibility that the detected cells were doublets of RAW 267 cells. 7-aminoactinomycin D (7-AAD), as well as isotype controls were used to exclude dead cells and the nonspecific binding of the monoclonal antibodies. Each group with no less than 10⁶ cells was gated for flow cytometric analysis.

Adult murine intestinal organoids in Matrigel/collagen type I and dFEnS were dissociated into single cells by incubating fragmented organoids in 50% v/v TryPLE Express (Gibco, #12605-101) in advanced DMEM/F12 for 10–15 min. These single cells were stained with FITC anti-mouse CD326 (Ep-CAM) (Clone G8.8, BioLegend, 1:500, #118207), PE anti-mouse Ly6A/E (Sca-1) (Clone D7, BioLegend, 1:5000, #108107), and propidium iodide (Immunostep, 1:1000, #PI) before flow cytometry analysis with FACS Melody (BD Biosciences, Franklin Lakes, N). Live single cells were gated to assess for Sca-1 and Ep-CAM.