Specific, homogenous bone marrow-derived macrophages (BMM) were obtained by ex vivo differentiation from mouse bone marrow progenitors in the presence of recombinant CSF1 as described previously ( Tushinski, R. J., I. T. Oliver, L. J. Guilbert, P. W. Tynan, J. R. Warner, and E. R. Stanley. 1982. Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy. Cell 28:71-81; Hume, D. A., and S. Gordon. 1983. Optimal conditions for proliferation of bone marrow-derived mouse macrophages in culture: the roles of CSF-1, serum, Ca2+, and adherence. J Cell Physiol 117:189-194). All mice were specific pathogen free (SPF), male and 6 to 8 weeks old at the time of bone marrow collection. The femoral and/or tibial bone was extracted, remaining muscle removed and the bone sterilised in 70% ethanol. The bone cavity was flushed with a 27-gauge needle using complete media (RPMI 1640 supplemented with 5 to 10% fetal bovine serum, 20 U/ml penicillin and 20 ug/ml streptomycin, 2 mM L-glutamine. The pooled bone marrow progenitors were cultured on bacteriological plastic at two to six plates per bone. Media was replaced on day five and cells re-seeded at day six onto tissue culture plastic. Cells were stimulated with lipopolysaccharide (LPS, Sigma) early on day seven and harvested over a time course (0, 1, 7h). Cells were harvested and RNA extracted using an RNeasy kit (Qiagen), according to manufacturer’s instructions.
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
