array scanning protocol
Microarray slides were washed and scanned with an Agilent Scanner, according to the standard protocol of the manufacturer.
nucleic acid hybridization to array protocol
The amplified cyanine 3-labeled cRNA samples were then purified using Promegas SV Total RNA Isolation System and hybridized to Agilent Whole Mouse Genome Microarrays, 8x60K according to the standard protocol of the manufacturer.
nucleic acid labeling protocol
Agilents Low Input Quick Amp Labeling Kit (Cy3)
Priming and activation of mast cells: Mature BMMC (>95% c-kit+Fc_RI+) were primed (sensitized) in the following way: cells were spun down, counted and re-suspended at the concentration of 1x106/ml in full mast cell medium containing 1ug/ml of anti-TNP IgE mAb (#557079, BD Pharmingen) overnight. 6 well plates were coated overnight at 4C with 5 ml of 500 ng/ml of TNP-BSA in PBS, or just PBS for non-activated samples. TNP-BSA-coated plates were washed 3x in PBS, then blocked for 2 hours in 3% detoxified BSA at RT followed by 3 more washes in PBS. IgE-primed BMMCs (20x106 in 5 ml medium) were incubated on PBS or TNP-BSA coated plates for 4 hours at 37C incubator. For non-activated conditions we generated 3 (WT) and 4 (KO) independent biological replicas. For activated conditions we generated 4 (WT) and 3 (KO) independent biological replicas.
Bone marrow cells were cultured at a concentration of 0.5x106/ml in medium [RPMI (Sigma), 10% heat-inactivated FBS (PAA), 5uM B-mercaptoethanol (Gibco), penicillin/streptomycin (Gibco), 2 mM Glutamin (Gibco)] supplemented with 5 ng/ml of recombinant murine IL-3 (Peprotech, UK). Once per week, in the course of 4-5 weeks, cells were spun down, counted and re-seeded at the density of 0.5x106/ml. The purity of the BMMC population was monitored by FACS and cells were only used for assays if the c-kit+Fc_RI+ mature BMMC constituted >95% of the total cultured cells.
nucleic acid extraction protocol
RNA was isolated from IgE-primed or activated mast cells using 1 ml Triazol.