Wheat Germ Agglutinin (WGA) Alexa Fluor® 594 Conjugate, Alexa Fluor® 594‐Phalloidin, Alexa Fluor® 488‐Phalloidin, and Abs to ZO‐1, occludin, and claudin‐5, were purchased from Life Technologies (Grand Island, UY); Claudin‐15 Ab was a gift from Dr Mikio Furuse (Kobe University, Japan); Claudin‐12 Ab from Santa Cruz Biotechnology (Santa Cruz, CA); Heparanase Ab from ProSpec (East Brunswick, NJ); and heparan sulfate proteoglycan Ab from US Biological (Marblehead, MA). Y‐27632, ML‐7, SB202219, and SP600125 were from EMD Millipore (Billerica, MA). TNF was from Peprotech (Rocky Hill, NJ). Broad‐ spectrum caspase inhibitors, z‐VAD‐fmk and boc‐D‐fmk, were from MP Biomedicals (Solon, OH).
Z vad fmk
Manufactured by MP Biomedicals
Sourced in United States
Z-VAD-fmk is a cell-permeable pan-caspase inhibitor that irreversibly binds to the catalytic site of caspase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Tumor necrosis factor (tnf), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Sp600125, by Merck Group (1 mentions)
Facsverse cytometer, by BD (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Rat igg, by Jackson ImmunoResearch (1 mentions)
Anti il 12, by BioLegend (1 mentions)
Anakinra, by Amgen (1 mentions)
Methaneseleninic acid, by Merck Group (1 mentions)
Bortezomib, by Santa Cruz Biotechnology (1 mentions)
Antibodies to actin, by Merck Group (1 mentions)
Streptavidin hrp, by Merck Group (1 mentions)
Mda mb 231 cell line, by American Type Culture Collection (1 mentions)
Bafilomycin a1, by Cayman Chemical (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Bafilomycin, by Merck Group (1 mentions)
Q vd oph, by MP Biomedicals (1 mentions)
11 protocols using z vad fmk
1
Immunofluorescence Staining of Tight Junction Proteins
2
Evaluating PE6 Protein's Apoptotic and TLR4 Modulation Effects
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RAW264.7 cells were seeded in 24-well plates at a density of 0.5 million cells/well. After 3 h of adherence, cells were treated with 5, 7.5, and 10 µg/ml of PE6 protein, and incubated for 24 h. Approximately 0.1 µM staurosporine (Sigma, USA) was used as a positive control. Approximately 20 µM pan-caspase inhibitor, ZVAD-FMK (MP Biomedicals, USA), was used as a negative control. Annexin V/PI staining was performed using a BD FACS apoptosis staining kit as per manufacturer protocol. Surface expression of TLR4 on Phorbol 12-myristate 13-acetate (PMA) differentiated (20 ng/ml of PMA for three days) THP1 cells was determined using (0.5 million) cells treated with different concentrations of PE6 (1 and 2 µg/ml). Cells were seeded in a 24-well culture plate. After differentiation, cells were treated with PE6 and harvested after 24 h and incubated with anti-human PE-TLR4 antibody. The samples were processed as per protocol provided by the supplier (BD Biosciences, San Jose CA, USA). LPS (500 ng/ml) treated cells were used as a positive control for TLR4 expression. Fluorescence intensity was measured using the BD FACSVerse cytometer (BD Biosciences). Untreated and heat HI PE6 treated samples were taken as negative controls. The data were analyzed by Flow Jo software (Tree Star Inc. USA).
3
Borrelia-Induced γδ T Cell Activation
4
Methaneseleninic Acid and Caspase Inhibition
5
Cell Line-Based Cytotoxicity Assay
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Human colorectal carcinoma cell line HCT116 p53+/+ and breast adenocarcinoma MDA-MB-231 cell line was from ATCC. HCT116 p53−/− were kindly provided by Prof. B. Vogelstein (Johns Hopkins University School of Medicine) [27] (link).
Cells culture medium DMEM was from PAN Biotech (Aidenbach, Germany) and fetal bovine serum (FBS) was from Hyclone (Cramlington, UK). The proteasome inhibitor bortezomib was obtained from Santa Cruz Biotechnology (Santa-Cruz, USA). General caspase inhibitor Z-VAD-FMK and inhibitor to lysosomal cathepsins E-64 were from MP Biomedicals (Eschwege, Germany), Bafilomycin A1 was from Cayman Chemicals (Ann Arbor, USA). Neutralizing antibodies to TRAIL death and decoy receptors were from Enzo Life Sciences (Farmingdale, USA) and R&D systems (Minneapolis, USA), respectively. FITC-conjugated antibodies to DR4, DR5, DcR1 and DcR2 receptor were obtained from Abnova (Walnut, USA), isotype control antibody was from Immunotech (Marcelle, France). For western blot biotinylated goat antibodies to TRAIL death and decoy receptors were purchased from R&D Systems (Minneapolis, USA), HRP streptavidin and antibodies to actin were from Sigma-Aldrich (St. Lotus, USA).
Cells culture medium DMEM was from PAN Biotech (Aidenbach, Germany) and fetal bovine serum (FBS) was from Hyclone (Cramlington, UK). The proteasome inhibitor bortezomib was obtained from Santa Cruz Biotechnology (Santa-Cruz, USA). General caspase inhibitor Z-VAD-FMK and inhibitor to lysosomal cathepsins E-64 were from MP Biomedicals (Eschwege, Germany), Bafilomycin A1 was from Cayman Chemicals (Ann Arbor, USA). Neutralizing antibodies to TRAIL death and decoy receptors were from Enzo Life Sciences (Farmingdale, USA) and R&D systems (Minneapolis, USA), respectively. FITC-conjugated antibodies to DR4, DR5, DcR1 and DcR2 receptor were obtained from Abnova (Walnut, USA), isotype control antibody was from Immunotech (Marcelle, France). For western blot biotinylated goat antibodies to TRAIL death and decoy receptors were purchased from R&D Systems (Minneapolis, USA), HRP streptavidin and antibodies to actin were from Sigma-Aldrich (St. Lotus, USA).
6
Antibody Preparation for Mouse RIPK3 and MLKL
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Antibodies; Rat-anti mRIPK3 and rat anti-mMLKL 8F6 (selected for affinity to residues 1–30 of mouse MLKL) and rat anti-MLKL 3H14 (link) (MLKL brace region) were produced in-house. Anti-Pro Caspase 8 (#4927) and GAPDH (#2113) were purchased from Cell Signaling Technology. Anti-mouse MLKL pS345 (ab196436) and anti-Actin (ab5694) were purchased from Abcam. Anti-VDAC (AB10527) was purchased from Millipore. Fc-hTNF was produced in house and used at a final concentration of 100 ng per mL. Recombinant mouse IFN-γ and β were purchased from R&D Systems (Minneapolis, MN, USA) Q-VD-OPh and zVAD-fmk were purchased from MP Biomedicals (Seven Hills, NSW, Australia). Smac mimetic also known as Compound A, and the caspase inhibitor IDN-6556 were a gift from TetraLogic (Malvern, PA, USA). Propidium iodide, doxycycline, and bafilomycin were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).
7
Quantification of Caspase-6 Activity
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Casp6 active sites were quantified using zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone, MP Biomedicals, Santa Ana, CA, USA)51 (link),64 (link). Casp6 activity was measured in Stennicke’s buffer (SB: 20 mM piperazine-N,N′-bis-(2-ethanesulfonic acid) pH 7.2, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonic acid, 10% sucrose)65 (link) using 40 µM Ac-VEID-AFC (Ac-Val-Glu-Ile-Asp-7-Amino-4-trifluoromethylcoumarin, Enzo Life Sciences, Farmingdale, NY, USA)64 (link) and 10-250 nM active site titrated Casp6. Reactions were initiated by Ac-VEID-AFC addition and followed for 20 min at 37 °C in a black clear bottom 96-well microplate (Costar, Corning, NY, USA) in Synergy H4 Hybrid instrument (BioTek, Winooski, VT, USA) with excitation/emission at 380/505 nm. Released AFC was calculated from AFC standard curve (0-25 µM). Casp6 VEIDase activity (released pmol AFC/min) was calculated from the initial linear reaction part. Ac-VEID-AFC hydrolysis initial velocities were measured with 10 nM active-site titrated Casp6 and 1–300 nM Ac-VEID-AFC. Initial velocities v versus substrate concentration [S] were fit to a rectangular hyperbola using Michaelis-Menten equation in GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
8
Caspase-6 Active Site Quantification
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9
Caspase-3/7 Activation and Apoptosis Assay
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H929 cells cultured in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) were pretreated with a pan-caspase inhibitor z-VAD-fmk (MP Biomedicals, Solon, OH; 50 µM) for 1 h before the addition of ABBV-467 for a further 6 h. Cells were then washed twice with Dulbecco’s phosphate-buffered saline and resuspended in staining buffer for determination of caspase-3/7 activation, Δψm, and annexin-V positivity using the Intellicyt MultiCyt 4-Plex Kit and high-content flow cytometry (Intellicyt, Albuquerque, NM).
10
Cell Culture and Compound Treatment Protocols for Apoptosis Assays
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NHL cell lines were obtained from the American Type Culture Collection or Deutsche Sammlung von Mikroorganismen und Zellkulturen and were cultured in Iscove's Modified Dulbecco's Media containing 10% human serum and 10 mM L -glutamine (all from Invitrogen Corporation, Carlsbad, CA, USA). All cell lines were tested for authenticity by short tandem repeat profiling and mycoplasm by the AbbVie Core Cell Line Facility. Cells were plated at a density of 0.25 × 106cells/well in six-well plates for apoptosis assays, at 0.1 × 106/ml for cell viability assays, and at 3 × 106per 10 cm2 petri dish for western blots. Navitoclax, venetoclax, A-1210477 and A-1155463 were dissolved in anhydrous dimethyl sulfoxide to a stock solution of 10 mM . Flavopiridol was dissolved in dimethyl sulfoxide at 1 mM . After overnight attachment, cells were treated for up to 48 h with vehicle alone, navitoclax, venetoclax, A-1155463, flavopiridol or A-1210477, or in the described combinations. Where indicated, cells were pre-treated for 60 min with z-VAD-fmk (50 μM ; MP Biomedicals, Santa Ana, CA, USA). Navitoclax, venetoclax, A-1155463 and A-1210477 were synthesized as described.20 , 31 (link), 32 (link), 33 (link) Unless otherwise indicated, all chemical reagents were obtained from Sigma Aldrich (St. Louis, MO, USA).
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