For cell proliferation analysis, cells were seeded in 96-well plates (2000 cells/well) and after 24 h they were treated with 2 nM TNF (Roche Applied Science, Indianapolis, IN, USA), or with 5, 10, 25, 50 or 100 ng/mL of TRAIL (SuperKillerTRAIL, Enzo Life Sciences, Farmingdale, NY, USA), or with 2 nM TNF plus 25 or 50 ng/mL of TRAIL for 60 h. The TNF concentration used corresponded to the one detected in epithelial lesions and that showed higher antiproliferative effect on keratinocytes as shown by us [5 (link),7 (link),21 (link),23 (link),55 (link)]. All treatments were performed in octuplicates. At the end of the treatment, 10 µL of Alamar Blue (Invitrogen, Frederick, MD, USA) were added per well and cells were incubated at 37 °C for 4 h. After this period, Alamar Blue’s reduction was monitored at 570 and 600 nm in an Epoch Microplate Spectrophotometer (Bio-Tek, Winooski, VT, USA). For RNA and protein expression analysis, organotypic cultures maintained for 9 days at the medium-air interface raft cultures were treated with 2 nM TNF or 25 ng/mL of TRAIL for 72 h.
Superkillertrail
Manufactured by Enzo Life Sciences
Sourced in United States, Germany
SuperKillerTRAIL is a recombinant protein that functions as a ligand for the TRAIL receptors. It induces apoptosis in a wide range of cancer cell lines. The product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Tumor necrosis factor (tnf), by Roche (1 mentions)
Alamar blue, by Agilent Technologies (1 mentions)
Tumor necrosis factor (tnf), by Enzo Life Sciences (1 mentions)
Etoposide, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Chetomin, by Enzo Life Sciences (1 mentions)
Mithras lb 940, by Berthold Technologies (1 mentions)
Caspase glo assay kit, by Promega (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
7 aminoactinomycin d 7 aad, by BioLegend (1 mentions)
Propidium iodide, by BioLegend (1 mentions)
Rnase a, by Roche (1 mentions)
Facscan, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Superfasligand, by Enzo Life Sciences (1 mentions)
12 protocols using superkillertrail
1
Influence of TNF and TRAIL on Cell Proliferation
2
Apoptosis Induction by Anti-APO-1, TRAIL, and Chemotherapies
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Cells were treated with different concentrations of anti-APO-1 antibodies,61 (link) SuperKiller-TRAIL (Enzo Life Sciences, New York, NY, USA), etoposide (Sigma-Aldrich, St Louis, MO, USA), doxorubicin (Sigma-Aldrich) or chetomin (Enzo Life Sciences) for different time periods as indicated in the figure legends. Apoptotic cell death was examined by the analysis of DNA fragmentation according to the method of Nicoletti as described previously.8 (link) Results are presented as the percent of specific DNA fragmentation using the formula: (percentage of experimental apoptosis−percentage of spontaneous apoptosis)/(100−percentage of spontaneous apoptosis) × 100.
3
Caspase Activation Monitoring Protocol
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Activation of caspases was monitored using a Caspase Glo Assay kit (Promega, Madison, WI) with a luminometer (Mithras LB940; Berthold, Bad Wildbad, Germany) in accordance with the manufacturer's instructions. A protein synthesis inhibitor, cycloheximide (Sigma-Aldrich) was used to suppress the activation of NF-κB followed by induction of cytokines. Anti-TRAIL neutralizing antibody (Oriental Yeast, Tokyo, Japan) was used for inhibition of TRAIL activity, and recombinant TRAIL (Super Killer TRAIL; Enzo Life Sciences, Farmingdale, NY) was used as a positive control for monitoring activation of caspases and activity of neutralizing antibody.
4
Apoptosis Regulation in T and B Cells
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Pacific Blue-conjugated anti-mouse CD3ε antibody (clone 145-2C11), PE-Cy7-conjugated anti-mouse CD19 antibody (clone 6D5), Fluorescein isothiocyanate (FITC)-conjugated annexin V, Propidium iodide (PI), and 7-amino-actinomycin D (7-AAD) were purchased from BioLegend (CA, USA). Alexa Fluor 488-conjugated anti-mouse CD4 antibody (clone RM4-5) was purchased from BD Biosciences. Rabbit anti-phospho-JNK antibody (clone 81E11), rabbit anti-JNK antibody, rabbit anti-phospho-c-Jun antibody (clone D47G9), rabbit anti-c-Jun antibody (clone 60A8), rabbit anti-cdc2 antibody, rabbit anti-β-actin antibody (clone 13E5), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody were purchased from Cell signaling technology (MA, USA). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (MO, USA). RNase A was purchased from Roche Diagnostics (Mannheim, Germany). SuperKiller TRAIL was purchased from Enzo Life Sciences (NY, USA).
5
Apoptosis Induction and Quantification
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To induce apoptosis, cells were stimulated with SuperFasLigand (100 ng/ml) or SuperKillerTRAIL (50 ng/ml) (Enzo Life Sciences) in the presence of 1 μg/ml of cycloheximide (CHX, Sigma-Aldrich). Overall apoptosis was measured by staining the cells with Annexin V (556422, BD Biosciences)/7 AAD (559925, BD Biosciences) according to the manufacturer's protocol followed by their analysis using a FACScan (BD Biosciences). The early necrotic cells were considered to be Annexin V negative but 7 AAD positive, the apoptotic cells were considered to be positive for both Annexin V and 7 AAD64 (link). The data were analyzed using the BD Cell Quest pro software (version 5.2.1, BD). Overall apoptosis was presented as the mean value ± SD (n = 3). The significance of differences between populations of data was assessed according to the Student's two-tailed test (*P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005).
6
Breast Cancer Cell Line Apoptosis Assay
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The human breast cancer cell lines were obtained from ATCC (MDA-MB-231ER-HER2- SKBR3HER2+ BT474 ER+HER2+) and CLS (MDA-MB-468 ER-HER2- MDA-MB-436 ER-HER2-) except the MCF-7 line which was a kind gift from Dr Julia Gee, Cardiff University. All cell lines were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10 % foetal bovine serum (FBS) (Invitrogen), and 1 % penicillin-streptomycin and L-glutamine mix (Invitrogen). Cells were maintained at 37 °C in 5 % CO2.
Recombinant soluble human TRAIL was purchased as super-killer TRAIL from Enzo Life Sciences. Unless otherwise stated , cells were treated with 20 ng/ml TRAIL for 18 h before subsequent assays. The pan-caspase inhibitor Z-Vad-Fmk was purchased from R and D systems and used at a concentration of 20uM. Cells were pre-treated with caspase inhibitor for 1 h prior to treatment with TRAIL. Leptomycin B was purchased from Sigma and used at a concentration of 0.1 ng/ml.
Recombinant soluble human TRAIL was purchased as super-killer TRAIL from Enzo Life Sciences. Unless otherwise stated , cells were treated with 20 ng/ml TRAIL for 18 h before subsequent assays. The pan-caspase inhibitor Z-Vad-Fmk was purchased from R and D systems and used at a concentration of 20uM. Cells were pre-treated with caspase inhibitor for 1 h prior to treatment with TRAIL. Leptomycin B was purchased from Sigma and used at a concentration of 0.1 ng/ml.
7
Magnetically isolated B cell stimulation
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Magnetically isolated B cells at the concentration of 2.5–5 × 105/mL were stimulated with CD40L and IL-21 as described (34 (link)), or with CpG ODN 2006 (Apara Biosciences, Germany) in Iscove's Modified Dulbecco's Medium (IMDM; ThermoFisher Scientific) supplemented with 10% FCS, insulin, apo-transferrin, non-essential amino acids, glutamine and glutathione as described earlier (35 (link)). TRAIL (SuperKillerTRAIL, Enzo Life Sciences), Fc-hFasL made as described (36 (link)), TRAIL-R1 blocking antibody (MA1-19025, clone DR-4-02, ThermoFisher Scientific), poly-caspase inhibitor Q-VD (R&D Systems), and Staurosporine (Sigma-Aldrich) were added at indicated concentrations.
The human Burkitt lymphoma cell line BJAB (purchased from Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) was cultured in IMDM supplemented with 10% FCS.
The human Burkitt lymphoma cell line BJAB (purchased from Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) was cultured in IMDM supplemented with 10% FCS.
8
Modulation of Cell Cytotoxicity Pathways
9
Induction of Necroptosis in Cancer Cell Lines
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The gallbladder adenocarcinoma cell line Mz-ChA-1 and the pancreatic adenocarcinoma cell lines A818-4 and Pt45P1 have been described [50 (link)-52 (link)]. The colorectal adenocarcinoma cell line HT-29 cells was originally obtained from the American Type Culture Collection. Mz-ChA-1, A818-4 and Pt45P1 cells were cultured in RPMI 1640 (Life Technologies, Darmstadt, Germany) supplemented with 10% v/v FCS, 10 mM glutamine and 1 mM sodium pyruvate. HT-29 cells were cultured in McCoy’s supplemented with 10% v/v FCS, 10 mM glutamine. All cells were kept in a humidified incubator containing 5% w/v CO2. Necroptosis was induced by addition of human recombinant TRAIL (SuperKillerTRAIL™, Enzo, Lausen, Germany) in combination with benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk; Bachem, Heidelberg, Germany) and CHX (Sigma-Aldrich, Deisenhofen, Germany) or HHT (Santa Cruz Biotechnology, Heidelberg, Germany). Nec-1s and NSA were purchased from Merck Millipore, Darmstadt, Germany.
10
Cytotoxic Treatments for Renal Cell Carcinoma
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RCC cell lines and HEK293T cells were grown at 37 °C in an atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum (FCS), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Transduced RCC cells were maintained in the medium described above supplemented with 2 μg/ml puromycin. For treatment, cells were exposed to the following substances dissolved in culture medium for defined periods of time: 0.1 to 10 μg/ml topotecan (Hycamtin®, GSK, Buehl, Germany), 100 ng/ml superkillerTrail (EnzoLifeScience, Lörrach, Germany) or 10 or 20 μM ABT263 (Navitoclax®, Selleckchem, Texas, USA) or 50 μM UO126 (Selleckchem). The corresponding negative controls were prepared with the appropriate solvent (PBS or DMSO).
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