Toluidine blue

Toluidine blue (Shanghai Yuanye Biological Technology Co., Ltd., Shanghai, China) staining was used to detect mast cells in connective tissue of the colon of rats. Under a light microscope, the mast cells cytoplasm was purple red, the nucleus was blue, and the cell shape was round or oval or irregular. Smaller cells had less cytoplasm and clear borders; larger cells had more cytoplasm, with unclear borders, and purple-red particles were scattered around the nucleus. Under the guidance of experienced pathologists, mast cells were counted in three randomly selected visual fields.

For CFU-F assay, MSCs extracted from osteo-organoids were directly cultured in 6-well plates for 5 days (Table 1). After washed using Dulbecco’s phosphate-buffered saline, the MSCs were fixed using 4% (w/v) paraformaldehyde for 30 minutes and then stained using toluidine blue (0.1%; Yuanye Biotechnology Co., Shanghai, China). After being washed using ddH₂O, the colony formation could be observed under an optical microscope (DMi8; Leica, Hessian, Wetzlar, Germany).
For flow cytometry, purified MSCs were cultured until they reached 80–90% confluence. After washed using Dulbecco’s phosphate-buffered saline and digested, MSCstheywerestained using fluorescent antibodies in flow tubes and then analysed by flow cytometry. The cell aliquots were incubated: fluorescein isothiocyanate-conjugated CD45 or phycoerythrin-conjugated CD44, CD29 and CD31 or Alexa Fluor 700-conjugated stem cell antigen-1 and CD11b or Allophycocyanin-conjugated CD105 and CD140a (BD Biosciences, San Jose, CA, USA). The incubation was carried out at 4°C for 30 minutes in the dark. The cells were resuspended in 300 μL cell staining buffer (BD Biosciences) after washing. The flow cytometric analysis, including the test of EdU assay cell proliferation, was operated on CytoFLEX LX flow cytometer (Beckman Coulter, Bria, CA, USA). The data were analysed using FlowJo X (Three Star, San Carlos, CA, USA).