Pac1-/- bone marrow-derived mast cells stimulated with Fc epsilon receptor crosslinking for 2 hours.
Treatment protocol
.Bone marrow was isolated from femurs and tibias of 8-12 week-old Pac1-/- mice. Cells were grown at 4 × 10^5 cells/ml in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (HyClone), 100 μg each of penicillin and streptomycin per ml, 2 mM L-glutamine (Life Technologies), 50 μM β-mercaptoethanol, 1% HEPES, pH 7.5 and 5 ng/ml of recombinant murine IL-3 (Peprotech) for 4-6 weeks at 37 oC in 5% CO2. By 4 - 6 weeks in culture, greater than 98% of cells were c-kit and FcεRI positive as assessed by fluorescein isothiocyanate (FITC)-labelled anti-c-kit (BD, Pharmingen) and anti-DNP IgE (Sigma) antibodies, respectively. Cell morphology was also assessed by cytospin and staining with giemsa and toluidine blue. To stimulate, cells were sensitised with anti-DNP IgE mAb (Sigma, 100 ng/ml) for 18 hours, washed twice to remove unbound IgE and stimulated with 20 ng/ml of DNP-HSA (Sigma) for 2 hours.
Extracted molecule
total RNA
Extraction protocol
Trizol was used for total RNA extraction. cRNA was prepared according to methods for small RNA samples (Baugh et al, Nucleic acids research 29:E29, 2001). 500-1000 ng of RNA was reverse transcribed to cDNA using a poly(T) primer containing a T7-(dT)24 (Geneworks, Australia).
Label
PE
Label protocol
cRNA was transcribed from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Farmingdale, NY).
Hybridization protocol
Fifteen micrograms of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer (40mM Tris-acetate (pH 8.1), 125mM KOAc, 30mM MgOAc). Hybridization cocktails were then made by adding fragmented cRNA, control cRNAs, grid alignment oligonucleotides and blocking reagents. These mixtures were hybridized for 16 hours to individual U133A and B genechips (Affymetrix, Santa Clara, CA) at 45 °C. Washing and staining of the hybridized arrays were performed using an Affymetrix Fluidics Station.
Description
Pac1-/- bone marrow-derived mast cells stimulated with Fc epsilon receptor crosslinking for 2 hours where cells were sensitized with IgE anti-DNP for 18 hours and receptors cross-linked with DNP-HSA antigen for 2 hours.
