CFs, CMs and cocultures of CMs and CFs (CMs + CFs) were randomly assigned to one of five groups (Fig. 1 a): 1) blank control group (group C), in which the cells were incubated under normoxic conditions in a CO2 incubator; 2) H/R group (group M), in which the cells were subjected to H/R as described above; 3) H/R + DEX group (group D), in which the cells were pretreated with 1 μg/ml DEX at 2 hours before H/R; 4) H/R + MCC950 group (group 950), in which the cells were treated with 1 μM MCC950 (Selleck, USA; a potent selective NLRP3 inhibitor) in the H/R period and the previous 2 hours; and 5) H/R + DEX + MCC950 group (group D + 950), in which the cells were treated with 1 μM MCC950 during DEX and H/R pretreatment.
Mcc950
Manufactured by Selleck Chemicals
Sourced in United States, China
MCC950 is a potent and selective inhibitor of the NLRP3 inflammasome. It functions by blocking the activation of the NLRP3 inflammasome, which is a key component of the innate immune response. The MCC950 compound has been used extensively in research settings to investigate the role of the NLRP3 inflammasome in various disease models.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Vx 765, by Selleck Chemicals (5 mentions)
Adenosine triphosphate (atp), by Merck Group (4 mentions)
Nigericin, by Merck Group (4 mentions)
Mitosox, by Thermo Fisher Scientific (3 mentions)
Mitotracker, by Thermo Fisher Scientific (3 mentions)
Z vad fmk, by Selleck Chemicals (3 mentions)
Nigericin sodium salt, by Cayman Chemical (3 mentions)
Elisa kit, by Jiangsu Meimian (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Bay11 7082, by Merck Group (2 mentions)
Nigericin, by InvivoGen (2 mentions)
Anti nlrp3, by Adipogen (2 mentions)
Recombinant murine m csf, by Thermo Fisher Scientific (2 mentions)
Protein g agarose, by Merck Group (2 mentions)
Recombinant human nlrp3, by Novus Biologicals (2 mentions)
Poly a t, by Merck Group (2 mentions)
Anti human caspase 1, by Cell Signaling Technology (2 mentions)
Glucose, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
67 protocols using mcc950
1
Cardioprotective Effects of DEX and MCC950
2
Diabetic Db/Db Mouse Model Study
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Seven-week-old diabetic male db/db (C57BLKS/J-leprdb/leprdb) mice and non-diabetic male db/m littermates (C57BLKS/J-leprdb/+) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were maintained in a room with controlled environment (23±3°C, 50%±20% humidity, 12-hr light/dark cycle) with free access to water and food. All procedures were carried out according to the approved Institutional Animal Care and Use Committee protocol of China Medical University (Number: 16095M). The mice were randomly divided into three groups (n=10 per group). For the MCC950 treated group (db/db+MCC950), db/db mice were treated with 10 mg/kg of MCC950 (Selleck Chemicals, Houston, USA) twice per week intraperitoneally from 8 weeks of age to 20 weeks of age. The control (db/m) and untreated groups (db/db) received an equal volume of vehicle (saline). The treatment lasted 12 weeks. Body weight (BW) was measured weekly, and fasting blood glucose levels were measured every 4 weeks. All mice were sacrificed at 20 weeks of age. All mice were anesthetized with pentobarbital, and blood samples were collected by orbital vein bleeding. Kidney weight (KW) was measured after cardiac perfusion as described previously.21 (link) Renal cortical samples were harvested for subsequent studies.
3
Murine Macrophage Differentiation and Activation
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BMDMs were isolated from bone marrow of C57BL/6 wild-type or Caspase-1−/− (Jackson Laboratory) female mice (7–8 weeks old) and differentiated for 7 days in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml) and streptomycin (0.1 mg/ml) and 20 ng/ml human M-CSF (R&D Systems). THP-1 (Cell Bank of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 medium with FBS and penicillin and streptomycin. Phorbol 12-myristate 13-acetate (100 ng/ml, 24 h) was used in differentiation of THP-1 into macrophages. The additives or inhibitors used in in vitro experiments are as follows: purified SAK (0.5 μg/ml), lipopolysaccharide (LPS, 0.2 μg/ml), ATP (5 mM), MCC950 (1 μM, Selleck), potassium channel blocker glibenclamide49 (link) (10 μM, Sigma), KCl (25 mM), NADPH oxidase inhibitor apocynin50 (link) (200 μM, Selleck), lysosome membrane stabilizer dexamethasone51 (link) (200 nM, Sangon Biotech), ROS scavenger NAC52 (link) (1 mM, Sigma), and NF-κB inhibitor BAY 11-7082 (10 μM, Sigma).
4
NLRP3 Inflammasome Activation Assay
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ATP, nigericin, LPS, flagellin and poly(deoxyadenylic-deoxythymidylic) acid sodium salt (poly (dA:dT)) were purchased from InvivoGen (San Diego, USA). DMSO and MG132 was obtained from Sigma-Aldrich (Munich, Germany). MCC950 was purchased from Selleck (Houston, USA). Anti-DYKDDDDK-Tag antibody was purchased from MBL (Beijing, China). Anti-Myc-Tag, anti-HA-Tag and anti-β-actin were purchased from Proteintech (Wuhan, China). Anti-NLRP3, anti-ASC, anti-caspase-1 were obtained from AdipoGen (San Diego, USA). Anti-IL-1β and anti-NEK7 were purchased from Cell Signaling Technology (Danvers, USA). Secondary HRP-conjugated antibodies used were anti-mouse IgG, anti-rabbit IgG (Cell Signaling Technology, Danvers, USA).
5
NEC mouse model with cognitive evaluation
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Experimental NEC mouse model was established as described previously with a little modification [13 (link)]. In brief, 7-day-old mice were fed by gavage with hyperosmolar formula [Similac Advance infant formula (Abbott Nutrition) and Esbilac canine milk replacer (PetAg) at a ratio of 2:1] every 3 h, meanwhile, exposed to transient hypoxia (5% O2, 10 min) and hypothermia twice daily for 4 days. In some cases, pups in the NEC group received an intraperitoneal injection of MCC950 (10 mg/kg, Selleck) once a day for 4 consecutive days (NEC+MCC950 group). In addition, age-matched and unstressed breastfed pups served as control group. Body weight was recorded daily throughout the course of experiment. On day 5, mice were sacrificed, and the brain as well as intestinal tissues was harvested for further analysis.
For the cognitive evaluation study, some pups in NEC and NEC+MCC950 groups were kept breast-feeding after NEC induction until weaned and then subjected to Morris water maze test at 28-day-old. Age-matched breastfed mice without treatment were regarded as control.
For the cognitive evaluation study, some pups in NEC and NEC+MCC950 groups were kept breast-feeding after NEC induction until weaned and then subjected to Morris water maze test at 28-day-old. Age-matched breastfed mice without treatment were regarded as control.
6
Macrophage Differentiation and Bacterial Infection
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Human acute monocytic leukemia (THP-1) cells (ATCC TIB-202) were grown in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; EveryGreen, TianhangBio, Hangzhou, China). Around 2 × 106 cells in each well were differentiated into macrophage-like cells by treatment with 100 nm phorbol-12-myristate-13-acetate (PMA) (Sigma, St Louis, MO, USA) overnight. The cells were infected with live P. gingivalis or S. mitis at a MOI of 1:0.5, 1:5, or 1:50 for 2, 6, or 24 h, which was selected due to good cell viability (Supplementary Figure 2 ). According to the results of dose-dependent experiments, MOI = 50 was selected for application during time course assays with sampling at 2, 6, and 24 h or 1, 2, 6, and 24 h. In some experiments, the cells were co-stimulated with 5 mM ATP (Sigma) during bacterial infection. To inhibit NLRP3 inflammasome, cells were pretreated with MCC950 (S7809, Selleck, CN) for 1 h with indicated doses before stimulation.
7
Lipid Accumulation Cellular Model
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For cell experiments, 270 mg of RYR powder was immersed and extracted with the mixture of water, ethanol, and DMSO (8:1:1, v/v/v, 2 mL) at room temperature for 2 h, the resulting solution was centrifuged, and the supernatant was filtered using 0.22 μm filter membrane (Millipore, USA) [38 (link)]. HepG2 cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultivated in RPMI 1640 medium (Hyclone, Logan, UT). To develop a cellular model of lipid accumulation, cells were stimulated with 20 µg/mL LPS (Solarbio, Beijing, China) for 3 h followed by incubation with or without RYR (337 µg/mL and 674 µg/mL), lovastatin (50 µM, Innochem, Beijing, China) or MCC950 (10 µM, Selleck, USA) for 24 h, subsequently, the cells were subject to 0.25 mM palmitate acid (PA) (Sigma, St. Louis, MO, USA)/1% BSA (Solarbio, Beijing, China) for another 24 h. The stock solution of 5 mM PA/5% BSA solution was prepared as described [39 (link)].
8
SARS-CoV-2 Infection in hACE2 Overexpressing Cells
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Lentiviral vector-mediated in vitro gene delivery was performed as previously described.45 (link) Briefly, hACE2-overexpressing THP-1 cells were cultured with medium containing 8 μg/mL polybrene (Millipore) and 5 × 105 infectious units of lentiviruses containing scrambled shRNA or NLRP3-specific shRNAs (target sequence for human: 5’-CCGTAAGAAGTACAGAAAGTA-3’). At 48 h post infection, cells were infected with SARS-CoV-2 as indicated. In brief, THP-1 cells overexpressing hACE2 were primed with 250 ng/mL Pam3Cys (InvivoGen) for 4 h and then incubated with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 for 24 h. No infection (Mock) and nigericin (20 μM; Millipore) were used as a negative control and a positive control for NLRP3 activation, respectively. The NLRP3 inflammasome inhibitor MCC950 (15 μM; Selleck) was administered 1 h later after viral infection.
9
Neuroprotective Effects of Astaxanthin in OGD/R-induced Cell Injury
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SH-SY5Y neuroblastoma cells were purchased from the Shanghai Institute of Cell Biology. Cells were cultured in high glucose RPMI-1640 medium containing 10% fetal bovine serum (Gibco, batch number: C11875500BT) and incubated at 37°C in 5% CO2. The following cell culture groups were established: the sham group, the OGD/R group, high-dose, medium-dose, and low-dose AST IV groups, the AC-YVAD-CMK (Sigma-Aldrich, USA, Batch No.: SML0429) group, the MCC950 (Selleck, USA, Batch No.: S7809) group, and the AST IV + ML385 group. After the cells were washed with DPBS, the OGD/R group was treated with 37°C preheated glucose-free DMEM, and air (95% N2 and 5% CO2) was continuously ventilated for 1.5 L/min for 6 min. After ventilation, the hypoxic culture chamber was closed. Cells were placed in the incubator for 2 h. After the treatment, the glucose-free DMEM (Gibco, USA, Batch No.: 21922567) was removed, and a complete medium was added. Then, cells were put into the incubator for 24 h for reoxygenation. The sham group was cultured in a complete culture medium. Cells in the AST IV intervention groups were cultured in a medium with different concentrations of AST IV (5, 10, 20, 40, or 80 μg/ml), cells in the AC-YVAD-CMK group were treated with AC-YVAD-CMK, and cells in the MCC950 group were treated with MCC950.
10
Selective NLRP3 Inhibition in Subarachnoid Hemorrhage
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MCC950, a selective inhibitor of NLRP3 [24 (link)], was obtained from Selleckchem (Houston, TX) and diluted in saline. Saline was used as vehicle control for all experiments. MCC950 (40 mg/kg) or an equivalent volume of vehicle was delivered via intraperitoneal injection 15 min post-SAH. For experiments ending at 24 h post-SAH, an identical dose of MCC950 or vehicle was administered again 12 h later. For experiments ending 5 days post-SAH, MCC950 or vehicle was administered again on days 1 and 3 post-SAH (Supplemental Fig. 1 C). Dosage and dosing strategy were selected based on previous studies targeting the vasculature and ischemic stroke [25 (link), 26 (link)].
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