Peripheral blood mononuclear cell‐derived human mast
cells
Human peripheral blood mononuclear cell‐derived mast cells were
generated as previously described by Folkerts et
al. 33 Briefly, peripheral blood mononuclear
cells were obtained from buffy coats of healthy blood donors and
CD34+ precursor cells were isolated using the EasySep
Human CD34 Positive Selection Kit (STEMCELL Technologies).
CD34+ cells were maintained for 4 weeks under
serum‐free conditions using StemSpan medium (STEMCELL Technologies)
supplemented with recombinant human IL‐6 (50 ng/mL; Peprotech), human
IL‐3 (10 ng/mL; Peprotech), and human Stem Cell Factor (100 ng/mL
Peprotech). Thereafter, the cells were maintained in IMDM Glutamax I
containing sodium pyruvate, supplemented with 0.1% 2‐mercaptoethanol,
0.5% BSA, 1% insulin‐ 175 transferrin selenium (all from Invitrogen),
ciprofloxacin (10 µg/mL; Sigma‐Aldrich), IL‐6 (50 ng/mL; Peprotech), and
human Stem Cell Factor (100 ng/mL; Peprotech). After 8‐12 weeks, PBCMCs
were tested for maturity by Giemsa or toluidine blue staining and
beta‐hexosaminidase release assays.