6 protocols
AccessionNameType
P-MTAB-34141
array scanning protocol
Microarray slides were washed and scanned with an Agilent Scanner, according to the standard protocol of the manufacturer.
P-MTAB-34140
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.
P-MTAB-34139
nucleic acid labeling protocol
Agilents Low Input Quick Amp Labeling Kit (Cy3)
P-MTAB-34137
treatment protocol
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.
P-MTAB-34136
growth protocol
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.
P-MTAB-34138
nucleic acid extraction protocol
RNA was isolated from IgE-primed or activated mast cells using 1 ml Triazol.