strain: C57BL/6 gender: male developmental stage: young adult tissue: interscapular brown adipose fraction: stromal-vascular
Treatment protocol
For cellular separation, dissected adipose tissues were minced and incubated in 2.0 ml 0.2% collagenase type II in collagenase buffer (25 mM KHCO3, 12 mM KH2PO4, 1.2 mM MgSO4, 4.8 mM KCl, 120 NaCl, 1.2 mM CaCl2, 5 mM glucose, 2.5% BSA, 1% Pen/Strep, pH 7.4) rocking for 50 min at 37°C with occasional resuspension. 10 ml 90% PBS were added and samples centrifuged 5 min at 200 g. The adipocyte fraction was removed from the top and filtered through 100 µm cell strainers (BD). Filters were washed with 10 ml PBS and sample centrifuged as above. The adipocyte fraction was spun 2 min at 200 g and buffer removed from below the adipocytes with a 40 mm 20 G needle. The SVF pellet from the initial centrifugation was aspirated, resuspended in 2 ml erythrocyte lysis buffer (154 mM NH4lC, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4) and incubated for 4 min. After addition of 2 ml PBS samples were filtered through 40 µm cell strainers. Filters were rinsed with 8 ml buffer and samples centrifuged for 5 min at 200 g.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cells using Trizol reagent (Invitrogen) and Phase Lock Gel tubes (5prime) according to the manufacturer's instructions. Final RNA samples were re-precipitated with NaOAc and ethanol and washed with 70% ethanol before analysis on a NanoDrop-2000 spectrophotometer and Bioanalyzer 2100 (Agilent).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 600 ng RNA using the One-Color Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Agilent Gene Expression kit for oligo microarrays (Agilent, order number 5188-5242, protocol G4140-90050).
