Mil 9

To test myeloid potential, cells were cultured in IMDM with L-glutamine (Invitrogen), 20% heat inactivated fetal calf serum (Gibco), penicillin/streptomycin (Invitrogen) and 10^-4M β-mercaptoethanol (Sigma), supplemented with 20ng/ml mSCF (Peprotech), 20ng/ml mIL-3 (Peprotech), 10ng/ml mGM-CSF (Peprotech), 50ng/ml mIL-5 (R&D Systems) and 50ng/ml mIL-9 (R&D Systems) at 37°C, 5% CO₂. Bulk cultures of LMPP and pre-GM were started with 100-20.000 cells per culture. Myeloid potential of single cells was tested by sorting single cells directly into Terasaki plates, unless stated differently in the text, each well containing 20μl medium. GMP cells were derived in vitro by culturing progenitors (pre-GM or LMPP) for 3 days with 100ng/ml mSCF and 10ng/ml mIL-3 in round bottom 96 well plates and FcγRII/III⁺ cells were in this experiment considered as GMPs. In vitro derived GMPs were manually plated at an average of 5 cells/well into 60 well Terasaki plates. For eosinophil favouring culture conditions, GMP cells (200 cells/culture) were grown with 20ng/ml mSCF, 10ng/ml mGM-CSF and 50 ng/ml Il-5 in round bottom 96 well plates. After 5 days, the cells were washed and put back in culture for another 2 days without mSCF and mGM-CSF. All cytospins were prepared with a Shandon cytospin at 1000 RPM with low acceleration, followed by May-Grünwald-Giemsa stain (VWR).