Journal: Frontiers in Immunology

Article Title: Exposure to Dengue Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Endothelial Dysfunction and Hemorrhage in Mice

doi: 10.3389/fimmu.2021.617251

Figure Lengend Snippet: DENV- and rEIII-induced RCD in endothelial cells. (A) HMEC-1 endothelial cells treated with vehicle and various doses of DENV and rEIII; the live or dead status of the cell populations were determined using Zombie-NIR Kit labeling and flow cytometry analysis. [rEIII 1× = 0.3 µM, 2× = 0.6 µM, 4× = 1.2 µM; DENV e(rEIII 1×) is a DENV level equivalent to 0.3 µM rEIII]. (B) RCD inducers, doxorubicin (DOX; apoptosis) (2.5 µg/ml), rapamycin (autophagy) (0.5 µM), erastin (ferroptosis) (10 µM), TNF-α (necroptosis) (2.5 ng/ml), and nigericin (pyroptosis) (3.5 µM) induced relatively simple RCD patterns (flow cytometry gating and calculation methods described in Figure S3 ). By contrast, DENV and rEIII induced multiple RCD pathways, in which pyroptosis was the main RCD response, accounting for approximately 50% of total RCD response. If we normalize the respective RCD by the population of death cells (dead cell population normalized to 100%), we can obtain a more similar RCD pattern in DENV and rEIII groups (C) . ** P < 0.01 vs. vehicle groups.

Article Snippet: Treatments (1 h) of cell death inducers were used as positive controls for various types of RCD (apoptosis: doxorubicin, 2.5 μg/ml, Nang Kuang Pharmaceutical, Taipei, Taiwan; autophagy: rapamycin, 250 nM, Sigma-Aldrich; ferroptosis: erastin, 10 μM, Cayman Chemical; necroptosis: tumor necrosis factor alpha [TNF-α], 2 ng/ml, BioLegend; pyroptosis: nigericin, 3.5 μM, ImmunoChemistry Technologies, Minnesota, USA; 30 min in PBS).

Techniques: Labeling, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Exposure to Dengue Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Endothelial Dysfunction and Hemorrhage in Mice

doi: 10.3389/fimmu.2021.617251

Figure Lengend Snippet: Protection of endothelial cells from rEIII-induced pyroptosis under treatment with Nlrp3 inflammasome inhibitors. Treatment with Nlrp3 inhibitor CSB (chondroitin sulfate B, 10 μg/ml), OLT1177 (10 µM) and caspase 1 inhibitor Z-WHED-FMK (10 µM) rescued rEIII-induced endothelial cell death (A) . Treatments with CSB, OLT1177 and Z-WHED-FMK rescued cell dead population ( B, C, F, G, J, K ; cells with RCDs). If we normalize the respective RCD % by the population of death cells ( D : dead cell population normalized to 100%), we found that CSB, OLT1177 and Z-WHED-FMK preferentially rescued pyroptosis (E) , but not the other tested RCDs (H, I, L, M) . n = 6, ** P < 0.01, significant suppression vs. vehicle groups.

Article Snippet: Treatments (1 h) of cell death inducers were used as positive controls for various types of RCD (apoptosis: doxorubicin, 2.5 μg/ml, Nang Kuang Pharmaceutical, Taipei, Taiwan; autophagy: rapamycin, 250 nM, Sigma-Aldrich; ferroptosis: erastin, 10 μM, Cayman Chemical; necroptosis: tumor necrosis factor alpha [TNF-α], 2 ng/ml, BioLegend; pyroptosis: nigericin, 3.5 μM, ImmunoChemistry Technologies, Minnesota, USA; 30 min in PBS).

Techniques:

Journal: Mediators of Inflammation

Article Title: GPR34 Stabilized by Deubiquitinase USP8 Suppresses Ferroptosis of ATC

doi: 10.1155/mi/5576056

Figure Lengend Snippet: GPR34 deletion promotes ferroptosis of ATC cells. (A, B) The CCK8 assay was used to determine the viability of BHT101 (A) and 8305C (B) cells with GPR34 knockdown after addition of the apoptosis inducer VCR, ferroptosis inducer RSL-3, autophagy inducer rapamycin, or pyroptosis inducer LPS + nigericin. (C, D) Representative images were presented of BHT101 (C) and 8305C cells (D) with GPR34 deletion treated with RSL-3. (E, F) The CCK8 assay was used to assess the responses of BHT101 (E) and 8305C cells (F) with GPR34 silencing to RSL-3 ± Fer-1. (G) IHC test on xenograft tumors was performed to identify the correlation between GPR34 and 4-HNE expression. (H, I) Lipid ROS (H) and GSH (I) levels in GPR34 deletion ATC cells. Ns, nonsignificant. p > 0.05, ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001.

Article Snippet: Apoptosis inducer VCR (S1241), autophagy inducer Rapamycin (S1039), and pyroptosis inducer LPS (S7850) + Nigericin (S6653) were provided by Selleck.

Techniques: CCK-8 Assay, Knockdown, Expressing

Journal: Cellular Signalling

Article Title: Synergistic activation of p53 by actinomycin D and nutlin-3a is associated with the upregulation of crucial regulators and effectors of innate immunity

doi: 10.1016/j.cellsig.2020.109552

Figure Lengend Snippet: The activation of pro-caspase-1 by proteolytic digestion is not detected in cells treated with actinomycin D & nutlin-3a (A + N) combination or those treated with camptothecin (CPT). A. Expression of pro-caspase-1 detected in A549 cells treated, as indicated, for 48 h. Protein was detected using the D7F10 antibody from Cell Signaling Technology. The arrow shows pro-caspase-1. The lower band is apparently off-target protein. B. The expression of pro-caspase-1 in A549 cells treated, as indicated, for 48 h. This blot was incubated with ab179515 antibody from Abcam. Both panels are results of different exposures of the same blot. This antibody is able to recognize the small subunit (p10) of active form of the enzyme. C. Expression level in whole-cell lysates of total p53, its form phosphorylated on Ser392 and the expression of IFI16 and PYCARD proteins involved in formation of inflammasomes. The PYCARD protein, known also as ASC, has four splicing isoforms, three of them can be detected by antibody used in this study, which detects epitope near carboxyl terminus of protein. Two upper bands of PYCARD (approximately 19 and 22 kDa) are activating inflammasome adaptors, while the bottom band is an inhibitory adaptor (it lacks PYRIN domain). D. Western blots on protein lysates from A549 cells: Con - mock-treated, Nig - exposed to 15 μM nigericin for 70 min, A + N - exposed to actinomycin D and nutlin-3a for 49 h and for 70 min with fresh medium, A + N + Nig - exposed to actinomycin D and nutlin-3a for 49 h and subsequently to nigericin (15 μM) for 70 min. The blot was probed with anti-cleaved Caspase-1 (Asp297) rabbit monoclonal antibody (D57A2) from Cell Signaling Technology (CST). The blot on the left was probed with anti-caspase-1 antibody (D7F10) from CST.

Article Snippet: Stock solution of nigericin sodium salt (20 mM, Tocris Bioscience, Minneapolis, MN, USA) was prepared in ethanol.

Techniques: Activation Assay, Expressing, Incubation, Western Blot

Journal: Inflammopharmacology

Article Title: Dexketoprofen enhances NLRP3 activation via ATPase activity after canonical stimuli

doi: 10.1007/s10787-025-01928-2

Figure Lengend Snippet: Effect of DXK alone and with inflammatory stimulation in human macrophages. A Representative images of western blot analysis of macrophages exposed to DXK 100 nM for 15 min and 1 nM, primed with LPS 1 μg/mL for 4 h stimulated with nigericin 10 µM for 30 min. B IL-1β concentration in the supernatant measured by ELISA. C LDH activity measured in the supernatant. D DeltaVision confocal images of human macrophages exposed to DXK 100 nM and 1 nM, primed with LPS and stimulated with nigericin. Macrophages were labeled with ASC (green) and DAPI (blue). Scale bars, 10 µm. Data are mean + SEM from n = 3 independent experiments. E The percentage of ASC speck-containing cells was quantified using the imaging software Fiji. For each individual experiment, the average number of ASC specks per nucleus was calculated and plotted. A total of 100 cells per condition were imaged and quantified. Data are mean + SEM from n = 3 independent experiments. Data were analyzed using a Student’s t test. ns = p > 0.05, *, a = p ≤ 0.05, **, aa = p ≤ 0.01. * represents treatment vs control and a represents treatment vs LPS-Nig

Article Snippet: Phorbol 12-myristate-13-acetate (PMA) (P8139-MG, Sigma Chemical Co., St. Louis, MO, USA), dexketoprofen (sc-357330, Santa Cruz Biotechnology, Santa Cruz, CA, USA), lipopolysaccharide (LPS) (L4391, Sigma Chemical Co., St. Louis, MO, USA), nigericin (sc-201518B, Santa Cruz, CA, USA), adenosine triphosphate (ATP) (sc-202040A, Santa Cruz, CA, USA), bovine serum albumin (BSA) (9048–46-8, Sigma Chemical Co., St. Louis, MO, USA), DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA), saponin (47,036, Sigma Chemical Co., St. Louis, MO, USA), bioBLUPrestainesProtein loader (GTPBM0003, Biorad Laboratories Inc., Hercules, CA, USA).

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Imaging, Software, Control

Journal: Inflammopharmacology

Article Title: Dexketoprofen enhances NLRP3 activation via ATPase activity after canonical stimuli

doi: 10.1007/s10787-025-01928-2

Figure Lengend Snippet: Combined effect of DXK and MCC950 in human macrophages when exposed to nigericin. A DXK docked to the NATCH domain of human NLRP3 (PDB:6NPY) using AutoDock Vina . B Chemical structure of DXK and zoom-in of the binding pocket with a score of -8.4 kcal/mol. Hydrogen bonds in blue. C ATPase activity measured by ADP-Glo Kinase Assay Kit using purified human NLRP3 exposed 15 min to vehicle, DXK 10 µM, MCC950 10 µM and both before adding purified ATP 250 µM. Data of enzyme activity were normalized to vehicle. D IL-1β concentration in the supernatant from macrophages exposed 15 min to DXK 100 nM, MCC950 10 µM and both before nigericin 10 µM 45 min. E LDH activity measured in the supernatant. Data are mean + SEM from n = 3 independent experiments. Data were analyzed using a Student’s t test. ns = p > 0.05, *, a = p ≤ 0.05, **, aa = p ≤ 0.01. * represents treatment vs control and a represents treatment vs Nig in C and D. * represents DXK vs control and a represents MCC950 vs DXK and vs Nig in E

Article Snippet: Phorbol 12-myristate-13-acetate (PMA) (P8139-MG, Sigma Chemical Co., St. Louis, MO, USA), dexketoprofen (sc-357330, Santa Cruz Biotechnology, Santa Cruz, CA, USA), lipopolysaccharide (LPS) (L4391, Sigma Chemical Co., St. Louis, MO, USA), nigericin (sc-201518B, Santa Cruz, CA, USA), adenosine triphosphate (ATP) (sc-202040A, Santa Cruz, CA, USA), bovine serum albumin (BSA) (9048–46-8, Sigma Chemical Co., St. Louis, MO, USA), DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA), saponin (47,036, Sigma Chemical Co., St. Louis, MO, USA), bioBLUPrestainesProtein loader (GTPBM0003, Biorad Laboratories Inc., Hercules, CA, USA).

Techniques: Binding Assay, Activity Assay, Kinase Assay, Purification, Concentration Assay, Control

Journal: Cell reports

Article Title: Lidocaine induces apoptosis in head and neck squamous cell carcinoma through activation of bitter taste receptor T2R14

doi: 10.1016/j.celrep.2023.113437

Figure Lengend Snippet: SCC47 cells were loaded with Fluo-4 and imaged for subsequent Ca2+ responses with bitter agonists. (A and B) SCC47 Ca2+ trace (A) and peak Ca2+ (B) responses with lidocaine in HBSS ± extracellular Ca2+. Peak fluorescence mean ± SEM with >3 experiments using separate cultures. No significant difference by t test. (C) SCC47 peak Ca2+ response with nigericin stimulation in the presence or absence of extracellular Ca2+ and following stimulation with 10 mM lidocaine in the absence of extracellular Ca2+. Significance by one-way ANOVA with Bonferroni post-test comparing nigericin response to nigericin responses in Ca2+-free (–Ca2+) and post-lidocaine stimulation. (D–G) SCC47 cells were transfected with pcDNA-4mt3cpv (D and E) or pcDNA-D1ER (F and G) 24–48 h before Ca2+ imaging. (D and E) SCC47 mitochondrial (pcDNA-4mt3cpv) peak (mean ± SEM) (D) and representative trace (E) CFP/CFP-YFP fluorescence Ca2+ responses with HBSS, lidocaine or denatonium benzoate; >3 experiments using independent cultures. Significance by one-way ANOVA with Bonferroni post-test comparing HBSS to each agonist. (F and G) SCC47 ER (pcDNA-D1ER) change (F) and trace (G) of CFP/CFP-YFP fluorescence Ca2+ responses with lidocaine or denatonium benzoate. Peak fluorescence mean ± SEM with >3 experiments using separate cultures. Significance by one-way ANOVA with Bonferroni post-test comparing lidocaine to each concentration of denatonium. (H and I) SCC47 representative image of pcDNA-4mt3cpv (H) and pcDNA-D1ER CFP/YFP (I) baseline localization. Scale bar, 30 μm. (J) mRNA expression of ER Ca2+ channels in SCC47 relative to UBC. (K) SCC47 peak Ca2+ responses with 0–30 mM RyR agonist caffeine; mean ± SEM with >3 experiments using separate cultures. Significance by one-way ANOVA with Bonferroni post-test comparing HBSS to each concentration of caffeine. (L) SCC47 peak lidocaine Ca2+ responses ±1 h before ryanodine inhibition. Fluorescence means ± SEM with >3 separate cultures. No significant difference by unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no statistical significance (or no indication).

Article Snippet: Nigericin (sodium salt) , Cayman Chemical , 11437.

Techniques: Fluorescence, Transfection, Imaging, Concentration Assay, Expressing, Inhibition

Journal: Cell reports

Article Title: Lidocaine induces apoptosis in head and neck squamous cell carcinoma through activation of bitter taste receptor T2R14

doi: 10.1016/j.celrep.2023.113437

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Nigericin (sodium salt) , Cayman Chemical , 11437.

Techniques: Virus, Recombinant, Reverse Transcription, DC Protein Assay, Western Blot, CRISPR, Control, Expressing, TaqMan Assay, shRNA, Software

Journal: Scientific Reports

Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

doi: 10.1038/s41598-019-53965-x

Figure Lengend Snippet: Febuxostat inhibits IL-1β secretion in mitochondrial ROS-independent and -dependent manners. ( a ) Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 2 h with nigericin or MSU. Supernatant and cell lysate were used for immunoblotting. Data are representative of two independent experiments in which the same data were obtained. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin or MSU. IL-1β in the supernatant was analysed by ELISA. ( c ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin or MSU. After stimulation, cells were loaded with DHR123. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. # p < 0.05, ## p < 0.01, #### p < 0.0001 versus non-stimulated control. ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus nigericin or MSU-treated group. ns, not significant.

Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

doi: 10.1038/s41598-019-53965-x

Figure Lengend Snippet: Febuxostat inhibits cell death in mitochondrial ROS-independent manner. ( a ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin. LDH activity in the supernatant was measured. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 6 h with nigericin. After stimulation, cells were stained with calcein for living cell and PI for dead cell. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. ### p < 0.001, #### p < 0.0001 versus non-stimulated control. ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus nigericin-treated group.

Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

Techniques: Activity Assay, Staining

Journal: Scientific Reports

Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

doi: 10.1038/s41598-019-53965-x

Figure Lengend Snippet: Restored intracellular ATP is involved in the inhibitory effects of febuxostat. ( a ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin. Intracellular ATP was measured by luminescence method. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin. After stimulation, cells were loaded with TMRE. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. # p < 0.05, ## p < 0.01 versus non-stimulated control. * p < 0.05, ** p < 0.01, *** p < 0.001 versus nigericin-treated group. ns, not significant. ( c ) Primed BMDMs were incubated 6 h with glucose-free medium containing 10 mM 2DG in the presence of vehicle or febuxostat. RPMI1640 medium containing 10 mM glucose was used as normal medium. Intracellular ATP was measured. ( d ) IL-1β in the supernatant was analysed by ELISA. Data are representative of two independent experiments performed in triplicate and shown as mean ± SD. *** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

doi: 10.1038/s41598-019-53965-x

Figure Lengend Snippet: Febuxostat restored intracellular ATP by activating salvage pathway. ( a ) Schema of purine metabolism. ( b ) Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with nigericin. Intracellular ATP, ADP and AMP were measured by HPLC method. Total adenylate was calculated. Intracellular uric acid level was measured. Data are representative of two independent experiments in which the same data were obtained and shown as mean ± SD. * p < 0.05, ** p < 0.01,*** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

Techniques:

Journal: Scientific Reports

Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

doi: 10.1038/s41598-019-53965-x

Figure Lengend Snippet: Febuxostat improves cellular bioenergetics. ( a ) Heatmap image of metabolome data. Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with nigericin. Cell extracts were used for metabolome analysis. Red indicates increased expression whereas green indicates decreased expression. ( b ) Illustration of mitochondrial respiratory capacity. ( c,d ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin. OCR was measured with the Seahorse XFp Extracellular Flux analyser. Basal respiration and ATP production were calculated by subtracting OCR value at 73.7 and at 34.3 min from OCR value at 14.6 min in ( c ), respectively. Data are shown as mean ± SD of four independent experiments in singlet. * p < 0.05, ** p < 0.01.

Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

doi: 10.1038/s41598-019-53965-x

Figure Lengend Snippet: Schema of the mechanisms underlying the inhibitory effect of febuxostat on NLRP3 inflammasome activation. ( a ) MSU mainly activates mitoROS-dependent pathway, while nigericin mainly activates an iATP loss-dependent pathway (Black arrows in bold). In particular, nigericin induces mitochondrial dysfunction, and iATP loss by the disturbance of oxidative phosphorylation, finally leading to IL-1β secretion and cell death. Concomitantly, iATP degrades into uric acid via purine degradation pathway. Red arrows indicate the effects of NLRP3 inflammasome activators. ( b ) In the case of stimuli with mitoROS production, such as MSU crystals, febuxostat inhibits IL-1β secretion through the inhibition of mitoROS production. In the case of nigericin stimulation, febuxostat inhibits IL-1β secretion by restoring iATP and improving mitochondrial function via the salvage pathway activation. Blue arrows indicate the effects of febuxostat on NLRP3 inflammasome activation pathways.

Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

Techniques: Activation Assay, Inhibition