Bone marrow cells from tibias, femurs and hip bones from 8-10 week old C57BL/6 mice were harvested, lysed with 1x ammonium chloride lysing reagent (PharMlyse BD cat# 555899) for 5 minutes and resuspended with RPMI medium (Mediatech, Inc.) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin G, 100 U/ml streptomycin, and 292 ?g/ml of l-glutamine, 10 mM Hepes (pH 7.4), 0.1 mM nonessential amino acid, 1 mM sodium pyruvate, and 50 uM 2-mercaptoethanol with SCF (50 ng/ml) and IL-3–conditioned medium (final concentration of IL3 was 25 ng/ml). During the first 2-3 weeks of culture the medium was changed every 3 days, and the cells were transferred to new dishes to separate them from adherent cells. Cells were cultured for another 6-8 weeks with media change once a week until BMMC reach full differentiation. Mature mast cells are determined by FACS confirmation of c-Kit and FceR1 expression in >95% of the cell population containing double positives for both receptors. WEHI-3 cells were obtained from ATCC and were used as a source of IL3 to supplement the BMMC growth medium . WEHI-3 cells were cultured in IMDM containing 1.5g/L sodium bicarbonate, 100U/ml pen/strep, 1% L-glutamine, 0.05 mM 2-mercaptoethanol, and 10% FBS.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cell pellets using the RNeasy kit (Qiagen, Valencia, CA, USA).
Label
biotin
Label protocol
5 ug total RNA was used in a standard Affymetrix single amplification protocol.
Hybridization protocol
standard Affymetrix procedures except for use of custom GNF chip washer
Scan protocol
standard Affymetrix procedures using GCS3000 scanner
