For the quantitation of extracellular IL-1β, neutrophils primed with TNF-α or incubated in buffer were stimulated for 8 h with MSU at 37°C, and IL-1β was quantified in the cell-free supernatant using a commercially available ELISA kit obtained from eBioscience (catalog no. 88-7010-22; San Diego, CA, USA).
Following incubation in resting buffer (R), neurons were stimulated with 90 mM K+ for 90 s, then incubated in resting buffer for 10 and 30 min. CTX-HRP, added either during or after the stimulus (A ), or selectively after the stimulus (B ), was used as an endocytic tracer.
No significant increase in CD4+ (0.14%) and CD8+ (0.61%) T cells were observed when cells from naïve mice or mice immunized with buffer only (data not shown) were stimulated with different species of proteins.
CHO cells expressing Kv7.2-W236L were stimulated with buffer containing 30 mM K+ (to activate the channels) in the presence of concentrations of (S -2 between 0.024 µM and 100 µM.
Neutrophils (0.25 × 10/ml) were stimulated with buffer or with 100 ng/ml phorbol 12-myristate 13-acetate (Sigma), in the presence of Amplex Red (0.5 µ m) and horseradish peroxidase (1 U/ml), in a black 96-well plate with a clear bottom (Greiner, Alphen a/d Rijn, the Netherlands).
After incubation with Krebs-Ringer's buffer in presence of coelenterazine, they were stimulated with 11 mM glucose in order to activate the Ca2+-mediated insulin pathway.
Neutrophils were stimulated with HMGB1 10 ng/ml or buffer for 30 min followed by stimulation with MPO-ANCA-positive IgG or PR3-ANCA-positive IgG, normal IgG or buffer control for 1 h, respectively.
48 hours after seeding, the cells were stimulated with standard Tyrode's buffer as control, 50 nM endothelin-1, and 100 µM norephinephrine, which are agonists for the endogenously expressed GqPCR endothelin receptors and for the α1-adrenergic receptor, respectively.
The next day, the cells were stimulated with agonists in 50 µl of 1× stimulation buffer for 30 min at 37°C and lysed for 10 min with 9 µl of lysis/detection buffer.
The cells were stimulated with agonists, harvested, and lysed in buffer containing 1% Nonidet-P40 supplemented with complete protease inhibitor 'cocktail' (Roche) and 2 mM dithiothreitol.
