4.2. Mast Cell Cultures

Mouse bone marrow-derived mast cells (BMMC) were differentiated from the marrow of tibias and femurs of S1pr4^+/+ and S1pr4^−/− littermate mice and cultured for at least 6 weeks in RPMI 1640 supplemented with 10% FBS, 1M HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, 4 mM l-glutamine, 1 mM sodium pyruvate, 50 μM 2-meraptoethanol, 20 ng/mL IL-3, 20 ng/mL stem cell factor (SCF) and non-essential amino acids, as described [22]. Under these conditions, the proportion of mast cells in culture increases overtime and by 4–6 weeks >98% of the cells are mast cells. Mast cell expand particularly after 20 days in culture, when already >85% of the cells are mast cells. The purity of mast cells in the cultures was monitored by assessing the percentage of cells expressing the receptor for SCF, CD117 (Kit) and the IgE receptor, FcεRI, by flow cytometry. Functional studies were conducted on cultures containing >95% double-positive mast cells as described [62]. The total number of mast cells was calculated as: (Absolute total cell count in the culture X percentage of mast cells (FcεRI⁺/CD117⁺))/100 for each time point.

Peritoneal mast cells (PDMC) obtained from the peritoneal lavage of these mice were expanded in culture for 2 to 3 weeks in the same culture media as BMMC [22,63,64].