Calcium oxalate

Cells were stimulated with 100 ng ml⁻¹ LPS for 3 hours (Ultra pure lipopolysaccharide from E. coli 0111:B4 strain, InvivoGen, San Diego, California, USA), followed by addition of a second stimulus for supplemental 6 hours. The second stimulus consisted either of calcium oxalate (100 μg ml⁻¹ from a 1 mg ml⁻¹ stock stored at 4 °C) (Sigma Aldrich, St. Louis, Montana, USA), ATP (5 mM from a prepared 100 mM stock solution stored at −20 °C) (InvivoGen, San Diego, California, USA) or monosodium urate crystals (300 μg ml⁻¹ from 5 mg ml⁻¹ stock solution stored at −20 °C) (InvivoGen, San Diego, California, USA). The selective P2X7 inhibitors A740003 or apyrase (both from Sigma Aldrich, St. Louis, Montana, USA) were applied 15 minutes prior to the addition of the second stimulus. A740003 was dissolved in pure DMSO and cells were treated with 100 μM A740003^{10 (link)} or the equivalent amount of DMSO alone (0.5%). apyrase was dissolved in RPMI and cells were treated with 10U apyrase ml⁻¹ or the corresponding amount of RPMI only. At the end of every series supernatants were collected and RIPA buffer (Sigma Aldrich, St. Louis, Montana, USA) containing complete EDTA-free protease inhibitor (Roche, Mannheim, Germany) was added to each well to gain whole cell lysates.