To investigate the effect of 3MA on the ATP-dependent IL-1β release, cells pre-treated for 1 hour with RPMI with or without 10 mM 3MA were stimulated for 4 hours with LPS (10 ng/ml or 1 µg/ml).
Cells starved in 100-mm dishes were stimulated for 45 minutes, harvested in cold PBS and collected by centrifugation at 1000 × g.
All genes were normalized to the housekeeping gene peptidylprolyl isomerase A. 3T3-L1 adipocytes, grown under 5.5 or 25 mM glucose conditions, and isolated adipocytes, incubated under 5.5 or 25 mM glucose conditions, were stimulated with 10 ng/mL of recombinant mice TNF-α for 24 hours under serum-free conditions.
Tissue sections of 6 mm of diameter were stimulated in 48 well plates and frozen in OCT.
Similar results were obtained when isolated adipocytes from mice incubated under the 5.5 and 25 mM glucose conditions were stimulated with 10 ng/mL TNF-α.
The cultures were subjected to continuous perfusion with imaging buffer (136 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) and were stimulated with a train of 300 action potentials that were delivered at 10 Hz (100 mA, 1-millisecond pulse width).
Synoviocytes (3.5 × 106 per 140 mm diameter Petri dish) were stimulated with SNP at 0.5 mM for 48 hours.
To accomplish this acupuncture needles (0.18 × 30 mm) were inserted into the GV20 and GV14 to the depth of approximately 3 mm, after which the acupoints were stimulated at an intensity of 1 mA and a frequency of 2 Hz for 20 min using Grass S88 electro stimulator (Grass Instrument Co., West Warwick, RI).
Cells were stimulated by 5 mM MgCl2 plus 1 mM EGTA (ethylene glycol-bis-β-aminoethylether-N,N,N'N'-tetraacetic acid) or 100 nM phorbol 12-myristate 13-acetate (PMA).
Cells were stimulated with either 50 mM KCl, 10 mM ATP (Sigma) or 10 mM CPA (cyclopiazonic acid; Sigma).
Intact segments of coronary artery were stimulated with 60 mM high K+ or 10 mM caffeine using the protocol described above for agonists, and the resulting Ca2+ transients measured in individual smooth muscle cells within the vessel wall.
