Protocols for Isolating Murine Mast Cells

Bone Marrow-derived Mast Cell (BMMC) was obtained following the protocol described by (30 (link)). Briefly, bone marrow cells were obtained from femurs and tibias of 6-8-week-old C57BL/6 female mice. Cells were maintained in RPMI-1640 supplemented with 10% FBS, 5 μM β-mercaptoethanol (Life Technologies, USA) and 2% antibiotic and antimycotic (Sigma, USA) (complete RPMI 1640 medium) plus 10 ng/mL of murine recombinant IL-3 (Peprotech, USA) and 10 ng/mL of murine recombinant stem cell factor (Peprotech, USA)). Non-adherent cells were transferred to fresh culture medium twice a week for 6−9 weeks. The purity of BMMC was ≥90% assessed by flow cytometry after staining of CD117 (clone: 2B8, BioLegend, USA; 0.25 μg/mL) and FcϵRI (clone: MAR-1, BioLegend, USA; 0.16 μg/mL) (Supplementary Figure 1A).
Peritoneum-derived mast cells (PMC), cells were obtained from peritoneal cavity of mice and cultured in complete RPMI-1640 medium plus IL-3 (30 ng/mL) and SCF (20 ng/mL). Non-adherent cells were transferred to fresh culture medium twice a week for 3−4 weeks. The purity of PMC was ≥90% assessed by flow cytometry (Supplementary Figure 1C).
All experiments followed institutional biosecurity and safety procedures. All animal experiments were approved by the Research Ethics Committee of the ENCB, IPN (ZOO-016-2019).