Peripheral blood mononuclear cells at a density of 2 × 106 cells/mL were treated with 100 ng/mL of the Toll-like receptor 4 (TLR4) ligand E. coli lipopolysaccharide (LPS; Invivogen, San Diego, CA) in the presence or absence of 1 mM SSZ (Sigma-Aldrich, St. Louis, MO), and incubated for 24 h at 37°C in 5% CO2. The concentration of SSZ and the incubation time were selected after evaluation of cellular toxicity, where <1% of PBMC mortality [evaluated by Trypan blue exclusion staining and Fixable Viability Dye eFluor 506 (eBioscience)] was obtained at the chosen condition (n = 5, data not shown). Subsequently, the cells were stimulated with PMA and ionomycin (at 50 and 500 ng/mL, respectively), and incubated for 12 h at 37°C in 5% CO2, all in the presence of 5 μg/mL of Brefeldin A and monensin, previous evaluation of cell viability with the Fixable Viability Dye eFluor 506. Finally, the cells were harvested, followed by surface and intracellular staining, as described above. In a fraction of individuals, PBMC were cultured with LPS plus SSZ in the presence or absence of the caspase-1 inhibitor Ac-YVAD-cmk or the pan-caspase inhibitor Z-VAD-FMK (at 0.25 and 5 μg/mL, respectively; both from InvivoGen), and the type of cell death was evaluated with the TACS Annexin V Kits (Trevigen), following the manufacturer's instructions.
Ac yvad cmk
Manufactured by InvivoGen
Sourced in United States
Ac-YVAD-cmk is a synthetic peptide that acts as a potent and irreversible inhibitor of caspase-1. It functions by forming a covalent bond with the active site of caspase-1, effectively blocking its enzymatic activity.
Automatically generated - may contain errors
Lab products found in correlation
Z vad fmk, by InvivoGen (4 mentions)
Mcc950, by InvivoGen (4 mentions)
Adenosine triphosphate (atp), by InvivoGen (3 mentions)
Lipopolysaccharides (lps), by InvivoGen (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Anti cd3 antibody, by BD (2 mentions)
Msu crystal, by InvivoGen (2 mentions)
Ultrapure lps, by InvivoGen (2 mentions)
Puno nlrp3, by InvivoGen (2 mentions)
Mouse caspase 1, by R&D Systems (2 mentions)
Human or murine il 1β elisa kits, by R&D Systems (2 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Tacs annexin 5 kit, by Bio-Techne (1 mentions)
E coli lipopolysaccharide lps, by InvivoGen (1 mentions)
Nigericin, by Merck Group (1 mentions)
Glybenclamide, by InvivoGen (1 mentions)
Phorbol 12 myristate 13 acetate pma, by InvivoGen (1 mentions)
Z wehd fmk, by R&D Systems (1 mentions)
Tlrl lrna40, by InvivoGen (1 mentions)
Protease inhibitor, by Roche (1 mentions)
20 protocols using ac yvad cmk
1
TLR4 Activation and Inflammatory Response
2
NLRP3 Inflammasome Activation Assay
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Nigericin, LPS from E. coli strain 0111:B4, and BFA were from Sigma-Aldrich. Phorbol 12-myristate 13-acetate (PMA), MSU, ATP, alum, glybenclamide, Ac-YVAD-cmk, MCC950 (kind gift from F. Niedergang) were from Invivogen. Primary polyclonal rabbit anti-FADD (2782), monoclonal rabbit IgG anti-NLRP3 clone D2P5E, monoclonal rabbit IgG anti-CASPASE-1 clone D7F20, polyclonal rabbit anti-CASPASE-4 (4450), monoclonal mouse anti-CASPASE-8 (1C12), and monoclonal rabbit IgG clone E1E3I anti-ASC antibodies were from Cell Signaling Technologies. Monoclonal mouse anti-α-Tubulin clone DM1A and monoclonal ECL horseradish peroxidase-conjugated-linked mouse anti-β-Actin clone AC-15 were from Sigma-Aldrich. Secondary antibodies used for immunoblot, ECL horseradish peroxidase-conjugated-linked donkey antibody (F(ab′)2 fragment) anti-rabbit IgG (NA9340V), and ECL horseradish peroxidase-conjugated-linked goat anti-mouse IgG1 (M32107), were from GE Healthcare and Caltag, respectively.
3
Transfection and Inhibition of HIV-1 LTR
4
BMDM Inflammasome Activation Assay
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A macrophage-based assay was adapted from previously described protocols to assess inflammasome activation in BMDMs [60 (link)]. Frozen BMDMs were thawed and plated in 24-well or 96-well tissue culture-treated, flat-bottom plates in complete DMEM (DMEM + 2mM L-glutamine + 10% FBS + 1% Pen/Strep) at a density of 1x106 or 2x105 cells per well, respectively. After plating, cells were left to rest at 37°C and 5% CO2 overnight. BMDMs were then primed using 1μg/mL Pam3CSK4 (InvivoGen) for 17 hours (h) or left untreated. Primed BMDMs were stimulated with DENV NS1, 5μM nigericin (Invivogen), or left untreated. After 24h, supernatants were spun down at 10,600 x g for 10 minutes, and the cell-free supernatant was collected. BMDM layers were washed twice with PBS and then lysed in RIPA buffer (150mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50mM Tris pH 7.4) supplemented with protease inhibitor (Roche). Supernatants and cell lysates were stored at -80°C until further analysis. For experiments involving inhibitors, the NLRP3 inhibitor MCC950 (InvivoGen) or caspase-1 inhibitor Ac-YVAD-cmk (InvivoGen) were added at indicated concentrations 30 min prior to treatment with DENV2 NS1 or nigericin.
5
Cell Death Inhibitors Protocol
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If not otherwise specified, one of the following cell death inhibitors was added to the cells: (10 μM) 1–2 h prior to cell stimulation with dsDNA: Z-VAD(OMe)-FMK (Cell Signaling), Z-DEVD-FMK (R&D Systems), MCC950 (Invivogen), Nec-1 (Abcam) or Ac-YVAD-cmk (Invivogen).
6
NLRP3 Inflammasome Activation in HCASMCs
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All cells were kept at 5% CO2 and 37 °C. HCASMCs were treated with isolated NLRP3-YFP inflammasome particles (3:1 NLRP3-YFP particles/ cell) for 4 h (internalization, gene expression, migration, western blot) and 24 h (gene expression, extracellular matrix production). For mechanistic studies, NLRP3 inhibitor MCC950 (1 µM, Invivogen), caspase-1 inhibitor Ac-YVAD-cmk (25 µg/ml, Invivogen) and NFκB inhibitor IKK-16 (2 µM, Selleckchem) were added to the media 30 min before treatment. Endocytosis inhibitor Cytochalasin D (4 µM, Invitrogen) was added 30 min before treatment with NLRP3-YFP particles and was washed off 3 times by washing with culture medium.
7
Rat H9C2 Cardiomyocyte Hypoxia-Reoxygenation
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Rat heart-derived H9C2 cells were purchased from ATCC and cultured using DMEM medium (Hyclone, Logan, UT, USA) containing 100 IU/ml of penicillin–streptomycin (Beyotime, Shanghai, China) and 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37°C in a 5% CO2 incubator. For H/R treatment, H9C2 cells were subjected to 16 h of hypoxia (O2:N2:CO2, 1:94:5) followed by 2 h of reoxygenation. To inhibit the activity of caspase-1, cells were treated with AC-YVAD-CMK (10 μg/ml, InvivoGen, San Diego, CA, USA). To suppress the activity of PI3K, cells were treated with 5 μM GDC-0941 (MedChemExpress, Monmouth Junction, NJ 08852, USA). All experiments were performed in 12-well plates, and cells were seeded at a density of 0.1 × 106 cells/well. Cells were collected for further analysis 24 h after indicated treatments.
8
Inflammatory Cytokine Signaling Pathway
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Polybrene, TRIzol and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). PMA (Cat#P8139), ATP (Cat#A7699), LPS (Cat#L2630), U0126 (Cat#U120), and Bay11–7082 (Cat#B5556) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ac-YVAD-cmk (Cat#inh-yvad) was ordered from InvivoGen (San Diego, CA, USA). PierceTM DSS (Cat#A39267) was ordered from Thermo Scientific (Waltham, MA, USA). IL-1β (human) ELISA Kit II (Cat# 557966) was from BD Biosciences (San Jose, CA, USA). Antibody anti-GAPDH (Cat# 60004-1-lg) was obtained from Proteintech (Wuhan, China). Antibodies anti-NLRP3 (clone ID: D4D8T), IL-1β (clone ID: D3U3E), and Caspase-1 (clone ID: D7F10) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies anti-ASC (clone ID: B-3) and anti-ASC (clone ID: F-9) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
9
Inhibiting Caspase-1 Activity in Mice
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To inhibit caspase-1 activity in vivo, mice received Ac-YVAD-cmk (8 mg/kg per time, once weekly for 8 weeks; InvivoGen, San Diego, California) in dimethyl sulfoxide (5% v/v) or dimethyl sulfoxide alone intraperitoneally.
10
Fibroblast Viability after Oxidative Stress
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To assess cell viability of fibroblasts following oxidative stress, human fibroblasts corresponding to the ages of 49 and 52 y/o were grown in culture and exposed to 25 μM hydrogen peroxide for 30 min. Then, the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) was used according to manufacturer’s instructions to evaluate the number of lysed cells as described in [6 (link)]. Another group of cells (52 years-old) was exposed to 25 μM hydrogen peroxide and 10 μg/ml of the caspase-1 inhibitor Ac-YVAD-CMK (Invivogen) prior to LDH release analysis. Ac-YVAD-CMK is a cell permeable, irreversible, selective inhibitor that prevents the cleavage of caspase-1, thus preventing its activation. Studies were replicated 5 times for all groups.
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