Cultivation and identification of mouse peritoneal and bone marrow-derived mast cells

Objective To explore the in vitro culture methods for oriented differentiation of peritoneal cells and bone marrow cells into high-purity mast cells, and to identify the function of these mast cells. Methods Peritoneal cells and bone marrow cells were isolated from the peritoneal cavity lavages and femur of C57BL/6 mice, and cultured with both interleukin-3 (IL-3) and stem cell factor for 2 and 4 weeks respectively. Light microscopy was performed to observe the morphology of these cells, toluidine blue staining to identify the degree of maturity of these mast cells, and flow cytometry to measure the expression of cell surface markers CD117 and FceRⅠα. After the stimulation with compound 48/80 at different concentrations, the degranulation rate of mast cells was counted under the microscope, and β-hexosaminidase release rate was measured by spectrophotometry. Results After 2- or 4-week culture, the mouse peritoneal and bone marrow cells all manifested as refractive suspension cells of uniform size. Toluidine blue staining showed violaceous metachromatic granules in the cytoplasm of the two kinds of cells. The proportions of CD117 or FceR Ⅰ α single-positive peritoneal and bone marrow-derived mast cells were all more than 95%, and the proportions of CD117/FceRⅠα double-positive peritoneal and bone marrow-derived mast cells were 97.68% ± 0.80% and 96.12% ± 0.76% respectively. The degranulation rates of mast cells in the 100- and 1 000-mg/L compound 48/80 groups significantly differed from those in the blank control group (all P < 0.01) . Compared with the blank control group, the β-hexosaminidase release rates significantly increased in bone marrow-derived mast cells in the 100-mg/L compound 48/80 group and peritoneal mast cells in the 10- and 100-mg/L compound 48/80 groups (P < 0.01 or 0.05) . Conclusion IL-3 and stem cell factor can co-induce the directed differentiation and proliferation of mouse bone marrow stem cells and peritoneal cells, so as to harvest high-purity mature degranulated mast cells, and lay a foundation for subsequent cell biology research. Key words: Mast cells; Myeloid progenitor cells; Induced pluripotent stem cells; Mice; Cells, cultured; Interleukin-3; Stem cell factor