Title: Affymetrix CEL analysis. Description:
Affymetrix Generic Hybridization
Title: Affymetrix CHP Analysis (ExpressionCall). Description:
This document describes Affymetrix recommended labeling protocols for use with GeneChip Exon Arrays.
nucleic acid extraction protocol
Monolayers of adherent bone marrow-derived macrophages were washed twice with PBS. TRIzol reagent (Invitrogen, San Diego, CA) was added directly to the adherent cells. The homogenate was scrapped off and passed to Eppendorf tubes. Chloroform was added to the homogenate (200ul of chloroform for every 1ml of TRIzol used). The samples were inverted and mixed. The samples were centrifuged to get phase separation and the upper aqueous phase was removed and precipitated to get total RNA. For precipitation, 0.5 ml isopropyl alcohol were used for every 1ml of TRIzol used. The samples were incubated at -20C overnight and centrifuged. The RNA pellets were washed with 75% ethanol and centrifuged again. The total RNA pellet was dissolved in milliQ RNAse-free water. (Parameters: Extracted product = total_RNA, Amplification = none)
Bone marrow-derived macrophages were obtained from six to ten-week-old C57BL/6 mice. Bone marrow precursors were cultured for 7 days in DMEM supplemented with 20% heat inactivated FCS and 30% L-cell conditioned medium as a source of M-CSF. (Parameters: time unit = seconds, temperature unit = C, media = DMEM)
sample treatment protocol
After 7 days, bone marrow-derived macrophages were incubated in DMEM-1%FCS +/- the LXR agonist GW3965 (2micromolar) for 18h and then stimulated with IFN-gamma (5ng/ml) or PBS for 6 hours.