Immature LC populations were derived from cord blood CD34+ hematopoietic progenitor cells (STEMCELL Technologies Inc., Vancouver, Canada, six different donors) as previously described (Lauron et al., 2014 (link); Coronel et al., 2015 (link)). LC activation was induced by exposure to 1500 IU/ml of GM-CSF and 10% FBS (US origin, Gibco, Life Technologies, South San Francisco, CA), or 1500 IU/ml of GM-CSF and 200 ng/ml of CD40L (Peprotech, Rocky Hill, NJ), or 1500 IU/ml of GM-CSF and 250 ng/ml of LPS (Sigma-Aldrich, St. Louis, MO), or 1500 IU/ml of GM-CSF, 200 ng/ml of CD40L, and 250 ng/ml of LPS, or 1500 IU/ml of GM-CSF, 10% FBS, 200 ng/ml of CD40L and 250 ng/ml of LPS, or 125 U/ml of TNF-α, 400 IU/ml of IL-1β, 1000 IU/ml of IL-6 and 5 nmol/ml of PGE2 (all from BioVision, Inc., Milpitas, CA), or 1500 IU/ml of GM-CSF, 10% FBS, 200 ng/ml of CD40L, 250 ng/ml of LPS, 125 U/ml of TNF-α, 400 IU/ml of IL-1β, 1000 IU/ml of IL-6, and 5 nmol/ml of PGE2. CMV strain TB40/E was propagated on human foreskin fibroblasts (HFF) as previously described (Hertel et al., 2003 (link)).
Tnf α
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Denmark, Canada
TNF-α (Tumor Necrosis Factor Alpha) is a key pro-inflammatory cytokine involved in the regulation of immune cells and inflammatory processes. It plays a central role in the inflammatory response and is implicated in various pathological conditions.
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Lab products found in correlation
Interleukin 6 (il 6), by Abcam (115 mentions)
Il 1β, by Abcam (99 mentions)
Gapdh, by Abcam (30 mentions)
β actin, by Abcam (24 mentions)
β actin, by Merck Group (21 mentions)
Bcl 2, by Abcam (21 mentions)
β actin, by Santa Cruz Biotechnology (19 mentions)
Pvdf membrane, by Merck Group (18 mentions)
β actin, by Cell Signaling Technology (17 mentions)
Ifn γ, by Abcam (17 mentions)
Bcl2 associated x (bax), by Abcam (16 mentions)
Cox 2, by Abcam (15 mentions)
Cleaved caspase 3, by Abcam (14 mentions)
Il 10, by Abcam (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Cleaved caspase 3, by Cell Signaling Technology (13 mentions)
Caspase 3, by Abcam (13 mentions)
Mcp 1, by Abcam (13 mentions)
α sma, by Abcam (13 mentions)
Mmp 9, by Abcam (11 mentions)
457 protocols using tnf α
1
Deriving and Activating Immature Langerhans Cells
2
Stimulation of HaCaT cells with IL-17A and TNF-α
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The HaCaT cell line were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). HaCaT cells were cultured in Dulbecco’s Minimum Essential Medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. All of the culture media were purchased from Sigma (St. Louis, MO, USA), and fetal bovine serum was purchased from Gibco (catalogue no. 16000-044; Grand Island, NY, USA). IL-17A and TNF-α were purchased from Abcam (IL-17A, ab9567; TNF-α, ab9642). Cells were cultured to 80% confluence and incubated with IL-17A (50 ng/ml) or TNF-α (10 ng/ml) for 24 h. Astilbin was purchased from Ourchem Sinopharm Chemical Reagent (CAS#: 29838-67-3, XW080058) and was dissolved in dimethyl sulfoxide (DMSO, Beyotime, ST038) at a concentration of 10 mM and stored at − 20 °C.
3
Fasting Metabolic Profiling in Mice
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Fed hormones/metabolites levels were determined by collecting tail blood from mice that were without food for 3 h. Fasted hormones/metabolites levels were assessed in mice provided only with water ad libitum and without food for the indicated period. Time at day at which blood was collected was the same between groups. Tail vein blood was assayed for glucose levels using a standard glucometer (Nova Biomedical). Plasma was collected by centrifugation in EDTA-coated tubes (Kent Scientific) and assayed for leptin (Crystal Chem. Inc., Downers Grove, IL), insulin (Crystal Chem. Inc.), TNF-α (Biovision) and IL-6 (Biovision) levels using the indicated commercially available kits.
4
Boehmeria nivea Extract Inhibits Mast Cell Activation
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Anti-DNP IgE, DNP-bovine serum albumin (BSA), dexamethasone, and Evans blue were purchased from Sigma (St. Louis, MO, USA). The RBL-2H3 cell line (KCLB-22256) was purchased from the Korean Cell Line Bank (Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and penicillin-streptomycin (PS) were purchased from Hyclone (Logan, UT, USA). The histamine, TNF-α, IL-6, IL-1β, and IL-4 ELISA kits were purchased from Biovision (Milpitas, CA, USA). Anti-phospho-p38 (Thr180/Tyr182), anti-phospho-ERK (L352), anti-phospho-JNK (T183/Y185), anti-p38, anti-ERK, anti-JNK, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Boehmeria nivea is an eco-friendly pesticide-free product, which was purchased from a farming association (Yeonggwang, Korea).
5
Fasted Metabolic Biomarker Profiling
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Fed hormones/metabolites levels were determined by collecting tail blood from mice that were without food for 3 h. Fasted hormones/metabolites levels were assessed in mice provided only with water ad libitum and without food for the indicated period. Time at day at which blood was collected was the same between groups. Tail vein blood was assayed for glucose levels using a standard glucometer (Nova Biomedical). Plasma was collected by centrifugation (1000 xg) in EDTA-coated tubes (Kent Scientific) and assayed using the indicated commercially available kits: β-hydroxybutyrate (Sigma), insulin (Crystal Chem. Inc.), TNF-α (Biovision), IL1-β (Biovision) and IL-6 (Biovision), S100A9 (R&D) and NEFA (Wako Chemicals).
6
Apoptosis Induction and Inhibition Assay
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The following primary antibodies were used: mouse anti-HA tag from Zhongshan Golden Bridge Biotechnology (Beijing, China); rabbit anti-GAPDH from Bioworld Technology (United States); rabbit anti-caspase-8 from Beyotime Biotechnology (Jiangsu, China). The following secondary antibodies were used: peroxidase-conjugated goat anti-mouse IgG (H + L) and peroxidase-conjugated goat anti-rabbit IgG (H + L) from Zhongshan Golden Bridge Biotechnology.
The pharmacological inhibitors were as follows: caspase-8 inhibitor Z-IETD-FMK from BioVision (United States); pan caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Ac-DEVD-CHO from Beyotime Biotechnology. The apoptosis inducer cycloheximide (CHX) and TNF-α were purchased from BioVision and Peprotech (United States) respectively.
The pharmacological inhibitors were as follows: caspase-8 inhibitor Z-IETD-FMK from BioVision (United States); pan caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Ac-DEVD-CHO from Beyotime Biotechnology. The apoptosis inducer cycloheximide (CHX) and TNF-α were purchased from BioVision and Peprotech (United States) respectively.
7
Immunohistochemical Analysis of Asthma Biomarkers
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In order to analyze the changes in the levels of asthma-related protein, immunohistochemistry was performed using antibodies such as IFN-γ (Santa Cruz, sc-74104), IL-12p40 (Santa Cruz, sc-57258), IL-4 (Santa Cruz, sc-73318), IL-5 (Santa Cruz, sc-7887), IL-13 (Santa Cruz, sc-1776), IL-6 (Santa Cruz, sc-1265), and TNF-α (BioVision, 3053R-100, Milpitas, CA, USA) and the dilution rate ranged from 1:100 to 1:200. The slides were incubated with a biotinylated pan-specific secondary antibody for 10 min and reacted with the streptavidin-peroxidase complex for 5 min (Vector Laboratories Universal Quick Kit, Burlingame, Canada). Signals were detected using 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution. Cells were counterstained with Mayer’s hematoxylin. Subsequently, the cells were imaged using Axioscope A1 (Carl Zeiss).
8
TNFα-induced nuclear and cytoplasmic RNA extraction
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1 × 107 fish hepatocytes were incubated with 50 ng/mL TNFα (BioVision) for 12 h and collected to extracted nuclear and cytoplasmic protein extraction using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). For nuclear and cytoplasmic RNA separation, 1 × 106 fish hepatocytes were collected and extracted using Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Canada) according to the manufacturer’s instructions. Quantitative real-time PCR was used to determine the expression of LDLR-AS, with U6 and GAPDH used as internal references of nuclear and cytoplasmic RNA.
9
NF-κB Activation Assay in HEK293 Cells
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The GloResponse™ NF-κB-RE-luc2P HEK293 cell line (Promega, Cat. # E8520, USA) was used to screen the effect of LHQK and its active components [29 (link)] on TNF-α (Biovision; 10 ng/mL) mediated nuclear translocation of NF-κB according to manufactures instruction. Cells were cultured in DMEM media with 10% fetal bovine serum and 1 × antibiotic–antimycotic (Gibco, Life Technologies) at 37 °C in a humidified incubator under 5% carbon dioxide. MTS and cytotoxicity assay kit (Sigma Aldrich) was used to assess cell viability, and the maxium non-toxic concentration for each active ingredient tested in Additional file 1 : Figure S1 was selected as the dosage for the NF-kB luciferase reporter assay. Luciferase reporter assay was performed as reoprted. Briefly, cells were seeded in a white glass-bottom 96-well plate (Thermo Scientific), and pre-treated with active ingredients of LHQK for 2 h before TNF-α was added to trigger NF-κB activation. Luciferase activity was analyzed with ONE-Glo™ luciferase assay reagent using GloMax multimode reader (TECAN, Switerland).
10
Immunohistochemical Analysis of Cytokine Expression
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Immunohistochemical analysis was conducted as described in our previous study [9] (link). Briefly, after getting rid of paraffin from tissues on the slides in order to eliminate endogenous peroxidase they were immersed in 3% hydrogen peroxide in methanol for 10 min. Sodium citrate buffer (0.1 M) was used for the antigen retrieval. In order to prevent non-specific binding normal horse serum was used, and then they were bounded with primary antibodies (diluted 1:100 to 1:200) including IFN-γ (Santa Cruz, sc-74104), IL-12p40 (Santa Cruz, sc-57258), IL-4 (Santa Cruz, sc-73318), IL-5 (Santa Cruz, sc-7887), IL-13 (Santa Cruz, sc-1776), TNF-α (BioVision, 3053R-100, Milpitas, CA, USA), and IL-6 (Santa Cruz, sc-1265) antibodies at 4 • C for 1 h. Using biotinylated secondary antibody, all slides were incubated for 10 min and reacted with streptavidin peroxidase complex for 5 min (Vector Laboratories Universal Quick Kit, Burlingame, Canada). Signals were obtained using 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution and they were stained with Mayer's hematoxylin. The results were obtained by an Axioscope A1 (Carl Zeiss) and to measure the positive cells we counted them in five randomly selected non-overlapping fields (×200 magnification) of three separately.
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