The RNeasy RNA extraction kit, with on column DNAse digestion (Qiagen) was used, according to manufacturer's protocol.
Label
Cy3
Label protocol
Starting with 500ng of total RNA, Cy3 labeled cRNA was produced according to manufacturer’s protocol.
Hybridization protocol
For each sample, 1.65ug of Cy3 labeled cRNAs were fragmented and hybridized for 17 hours in a rotating hybridization oven.
Scan protocol
Slides were washed and then scanned with an Agilent Scanner.
Description
Intra-peritoneally inject 2 ml of 4% Brewer’s thioglycolate to mouse (5.8 mg thioglycolate/150 mg agar/200cc H20) and euthanize mouse 96 h later. Wash abdominal cavity with 10 ml of ice cold sterile PBS (endotoxin free) twice to remove cells from peritoneum. Keep cells on ice. 2. Spin down cells 1,000 RPM, 6 min, 4°C. Wash two times with sterile cold PBS-CMF (10-30 mls), decanting. Resuspend in DMEM+pen/strep+0.1% FBS. Count cells, and plate 1.5 million cells/well in 1 ml in a 12 well plate (can plate as low as 1.1 million if necessary, but keep consistent between cell types). Let settle for at least 2 h before changing media to cholesterol-loading media (1ml/well). Make fresh cholesterol-loading media every time: DMEM (0.1% FBS)/2mg/ml BSA (low LPS)/10ug/ml cholesterol, adding cholesterol at the end (after the FBS and BSA) while swirling. Let media swirl on rocker for 5 min. before using to ensure cholesterol is completely in solution. Incubate overnight. Next day: Wash 2x with warm sterile 1x PBS-CMF. Change media to DMEM/penstrep/2mg/ml BSA (low LPS)/25µg/ml polymyxin B (1ml/well). Incubate 30 min. before performing treating with ApoA-I (10 ug/ml) for 2 h.
Data processing
Data were obtained and processed with the Agilent Feature Extraction Software (v9.5). The resulting data were imported into Rosetta Resolver (v7.1) and log10 intensities were exported.
