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.
Tumor necrosis factor (tnf)
Manufactured by Enzo Life Sciences
Sourced in United States
TNF is a recombinant protein commonly used in research applications. It functions as a pro-inflammatory cytokine.
Automatically generated - may contain errors
Lab products found in correlation
Stempro accutase, 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)
Superkillertrail, by Enzo Life Sciences (1 mentions)
Alamar blue, by Agilent Technologies (1 mentions)
Tumor necrosis factor (tnf), by Thermo Fisher Scientific (1 mentions)
Rat tail collagen type 1, by Merck Group (1 mentions)
Egm 2mv bulletkit, by Lonza (1 mentions)
α actin, by Santa Cruz Biotechnology (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
α myc, by Merck Group (1 mentions)
α ripk1, by BD (1 mentions)
α casp8, by Santa Cruz Biotechnology (1 mentions)
Hyaluronic acid, by R&D Systems (1 mentions)
Lysis buffer, by Kurabo (1 mentions)
Creatinine, by Enzo Life Sciences (1 mentions)
Creatinine, by Cayman Chemical (1 mentions)
Interleukin 6 (il 6), by Enzo Life Sciences (1 mentions)
Mmp 1, by MyBioSource (1 mentions)
7 protocols using tumor necrosis factor (tnf)
1
Influence of TNF and TRAIL on Cell Proliferation
2
Isolation of EVs from hCMEC/D3 Cells
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hCMEC/D3 human cerebral microvascular endothelial cells (Merck Millipore, Darmstadt, Germany) were seeded onto rat tail collagen type I (Merck Millipore) coated flasks and maintained in complete endothelial cell growth medium-2 (EGM-2MV BulletKit, Lonza, Basel, Switzerland) at 37°C in 5% CO2. Cells were grown until 70–80% confluency in six T-225 flasks. They were then washed three times with phosphate-buffered saline (PBS) solution containing calcium and magnesium, and incubated at 37°C with 32 ml of EGM-2MV containing 5% of heat-inactivated exosome-depleted foetal bovine serum (Gibco/Thermo FisherScientific, Waltham, MA, USA) for 24 h. Three flasks were stimulated with 10 ng ml–1 TNF (Enzo Life Sciences, Farmingdale, NY, USA). After 24 h of incubation, the supernatant was collected for isolating the EVs. TNF at 10 ng ml–1 was used since it was previously shown that this concentration does not induce endothelial cell apoptosis.[32 (link)] Cells were detached using Stempro Accutase (Gibco), washed four times with PBS, pelleted and stored at −80°C. This workflow was repeated twice, resulting in six flasks/samples of cells per condition (± TNF) and two samples of EVs per condition since the supernatants from three flasks were pooled as described below.
3
Cloning and Mutagenesis of Constructs
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Constructs were generated by PCR and cloned into pAc, pMT, pcDNA3 (Invitrogen) or pEF6 and verified by sequencing. Specific point mutations were generated using site-directed mutagenesis with Pfu Turbo polymerase (Stratagene). The Ub-sgg10 fusion construct was generated by PCR using Easy-A polymerase and cloned into pMT-V5/His. The following antibodies were used: α-MYC (Sigma, M5546, 1:2,000), α-HA (Roche, 11867423001, 1:2,000), α-V5 (Serotec, MCA1360, 1:2,000), α-RIPK1 (BD Biosciences, 610459,1:1,000 for western blotting (WB) and 1:50 for immunofluorescence studies), α-Actin (Santa Cruz, sc-1615, 1:4,000), α-MYO7A (Developmental Studies Hybridoma Bank, 138-1-s, 1:1,000 for WB and 1:50 for immunofluorescence studies), α-CASP8 for WB (MBL, M032-3, 1:5,000), α-CASP8 to detect cleaved CASP8 (for human: R&D, AF1650, 1:2,000; for mouse: Cell Signaling Technology, 9429, 1:1,000), rabbit and goat α-CASP8 (Santa Cruz Biotechnology, sc-7890 and sc-6136, both 1:50 for immunofluorescence studies), CF488A-donkey α-mouse IgG (Biotium, A21202, 1:1,000), CF633-donkey α-rabbit IgG (Biotium, 20125, 1:1,000) and CF633-donkey α-goat IgG (Biotium, 20127, 1:1,000). SM (SM164) was a gift from Shaomeng Wang (University of Michigan) and TNF was obtained from Enzo Life Sciences. Uncropped WBs are shown in Supplementary Fig. 5 .
4
Plasma and Tissue Biomarker Analysis in Mice
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Blood was collected from the hearts of mice at the determined end points and centrifuged at 3000 rpm to isolate the plasma by a microvolume high-speed cooling centrifuge MX-201 (TOMY, Tokyo, Japan) and stored at −20 °C. The dorsal skin, livers, and kidneys were excised and homogenized with lysis buffer (Kurabo, Osaka, Japan), and then, the tissue lysate was centrifuged at 10,000 rpm. Plasma concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, creatinine, hyaluronic acid, endostatine, glutamate oxaloacetate transaminase (GOT), glutamic acid pyruvate transaminase (GPT), matrix metalloproteinase (MMP)-1, histamine, and angiopoitin 1 and 2 were measured using appropriate ELISA kits according to manufacturers’ instructions (IL-6, Enzo Life Sciences, Farmingdale, NY, USA; TNF-α, hyaluronic acid, and angiopoietin 2, R&D Systems, Minneapolis, MN, USA; MMP-1, MyBioSource, San Diego, CA, USA; histamine, Bertin Pharm, Montigny-le-Bretonneux, France; creatinine, Cayman, Ann Arbor, MI, USA; Endostatine, BioMedica, Vienna, Austria; angiopoietin 1, EIAAB Science, Wuhan, China; GOT and GPT, Wako, Osaka, Japan; ROS, Cell Biolabs Inc., San Diego, CA, USA). Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
5
Astrocyte Activation by Inflammatory Stimuli
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Immortalized human fetal astrocytes (Applied Biological Materials Inc.) were seeded onto rat tail collagen type I-coated plates (15 μg/ml, Merck Millipore) at 5000 cells/cm2 and maintained in Prigrow IV medium (Applied Biological Materials Inc.) supplemented with 10% heat-inactivated FBS (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37 °C in 5% CO2. The medium was replaced every other day. At 80% confluence, cells were treated, in triplicates, with TNF (Enzo Life Sciences) (100 ng/ml), IL-1β (Enzo Life Sciences) (100 ng/ml), and ultra-pure LPS-EK (InvivoGen) (10 μg/ml) for 24 h or as indicated. Cells were detached using StemPro Accutase (Gibco), washed three times with ice-cold phosphate-buffered saline (PBS, Gibco), and dry-stored at − 80 °C.
6
HT1080 Cell Viability Assay
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A total of 5 × 103 HT1080 cells were seeded per well of a 96-well plate, overnight. Indicated siRNA were delivered for 48 h, before overnight treatment with DMSO, 10 ng/mL TNF (Enzo), and 100 nM SM164 (Insight Bio) individually or in combination. Viability was then assessed by CellTiter-Glo assay (Promega) according to manufacturer’s instructions, using a Victor ×5 HTS microplate. Graphs and statistical analysis were performed using GraphPad Prism V9.0. Standard two-way ANOVA tests were used to assess statistical differences in viability assays.
7
Apoptosis Induction Reagent Procurement
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Novartis provided LCL161 (18) . SM-164 was provided by S. Wang (University of Michigan, USA) (54) . Birinapant was obtained from Tetralogic Pharmaceuticals (55) . OICR720 was synthesized by the Ontario Institute for Cancer Research (OICR) (56) . AT-406, GDC-0917, and AZD-5582 were purchased from Active Biochem. TNF- was purchased from Enzo. IFN- was obtained from PBL Assay Science. All siRNAs were obtained from Dharmacon (ON-TARGETplus SMARTpool) or Ambion (Silencer Select for IFR1). High-molecular weight poly(I:C) was obtained from InvivoGen. VP16, staurosporine, and cycloheximide were obtained from Sigma.
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