mouse id#: 9 cell type: Bone marrow derived cells treatment: OVA immune Complex timepoint: 4hr
Treatment protocol
For ovalbumin immune complexes, endotoxin-free ovalbumin (EndoGrade Ovalbumin, Hyglos GmbH) was opsonized with a polyclonal rabbit anti-ovalbumin antibody (Sigma-Aldrich) at 37C for 1h. Subsequently BMDMs were treated with OVA-IC for 4 or 14 hrs or left untreated.
Growth protocol
Murine bone marrow was flushed form femurs and tibias, plated in regular petri dishes and supplemented with 100 ng mL-1 macrophage colony-stimulating factor (M-CSF, Peprotech) in complete medium (10% Hyclone FBS, 1% penicillin/streptomycin in RPMI 1640) twice over 5 days. On day 6, macrophages were transferred to tissue culture plates for stimulation.
Extracted molecule
total RNA
Extraction protocol
Cells were lysed and RNA extracted using Ambion pure link RNA minikit as per kit instructions.
Label
biotin
Label protocol
RNA was assessed for concentration and quality using a SpectroStar (BMG Labtech, Aylesbury, UK) and a Bioanalyser (Agilent Technologies, Cheadle, UK). Microarray experiments were performed at Cambridge Genomic Services, University of Cambridge, using a species specific Gene 2.1 ST Array Plate (Affymetrix, Wooburn Green, UK) in combination with WT PLUS amplification kit (Affymetrix) according to the manufacturer’s instructions. Briefly, 100ng Total RNA was amplified along with inline PolyA spike in control RNA, using the WT PLUS amplification kit (Affymetrix). Successfully amplified samples were labelled using the GeneChip WT terminal labelling kit (Affymetrix) using the in line hybridization controls.
Hybridization protocol
GeneTitan Hybridization, Wash and Stain kit (Affymetrix)
Scan protocol
Plate arrays were processed on the GeneTitan instrument (Affymetrix)
