During termination by intracardiac perfusion, a blood sample was taken from each mouse. Prior to infusion of PBS, a 20G needle was inserted into the caudal vena cava from which blood was collected. The blood was allowed to clot at room temperature for ~15 min. The samples were centrifuged at 735g at 4°C for 10 min and the supernatant was collected and stored at −20°C until processing. Interleukin 6 (IL-6) and tumor necrosis factor (TNF) were quantified using a Mouse IL-6 ELISA Kit (CAT# KMC0061; Invitrogen) and Mouse TNF ELISA Kit (CAT# 88–7324-22; Invitrogen), respectively. Sample/standard preparation and the ELISAs were performed according to the company’s recommended protocol.
Mouse tnf elisa kit
Manufactured by Thermo Fisher Scientific
The Mouse TNF ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of mouse tumor necrosis factor (TNF) levels in cell culture supernatants, serum, and plasma samples.
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Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Mouse il 6 elisa kit, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Procartaplex multiplex immunoassay kit, by Thermo Fisher Scientific (1 mentions)
Bio plex instrument, by Bio-Rad (1 mentions)
Ultra turrax t25, by IKA Group (1 mentions)
Active β catenin, by Cell Signaling Technology (1 mentions)
Total β catenin, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Bio plex pro mouse cytokine 23 plex kit, by Bio-Rad (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Bicinchoninic acid bca assay kit, by Thermo Fisher Scientific (1 mentions)
Mouse tnf alpha elisa ready set go kit, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
12 protocols using mouse tnf elisa kit
1
Quantification of Cytokines in Mouse Blood
2
Cytokine and Chemokine Profiling in Murine Lungs
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Serum was collected and whole lungs were harvested on days 0, 2, and 4 p.i. Lungs were disrupted using a tissue homogenizer (Ultra-Turrax T25; IKA Works, Inc., Wilmington, NC) in Cell Lysis Buffer (eBioscience). Lung homogenates were centrifuged at 2000 rpm for 10 mins, and supernatants were collected. The protein levels of 20 different cytokines and chemokines in the lung and serum were determined using a ProcartaPlex Multiplex Immunoassay kit (eBioscience) according to the manufacturer’s instructions. The assay was run on a BioPlex instrument (Bio-Rad, Hercules, CA). Lung and serum IFN-γ levels were determined by ELISA as previously described (eBioscience) [68 (link)]. Lung TNF levels were determined using a mouse TNF ELISA kit (Invitrogen) according to manufacturer’s instructions.
3
HMGB1 Binding and TNF Production Assays
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Polystyrene microplates (Costa, 3590) were coated with recombinant HSP90AA1 (ATGen, ABIN806289) diluted in carbonate-bicarbonate buffer (pH 9.6) at 4°C for 18 h and then washed three times with PBST. The wells were blocked with PBST containing 3% BSA at 37°C for 2 h. Recombinant HMGB1 was serially diluted in 3%-PBST and added to the wells for 1 h at 37°C. After washing, the anti-HMGB1 antibody was added to each well for incubation for 1 h at 37°C. Horseradish peroxidase-conjugated goat anti-rabbit IgG was added for 30 min at 37°C. Next, 3,3,5,5ʹ-tetramethylbenzidine (TMB; Invitrogen, 002023) substrate was added, and the reaction was stopped by adding 3 N HCl. The optical density was read at 450 nm.
To measure TNF production, RAW264.7 cells were pretreated with 100 nM Wort, 20 nM Baf, 25 μM CQ, and 2 μM GW4869 for 2 h and then treated with 250 ng/mL LPS or 50 μM H2O2 for 24 h. The mouse TNF level was measured using a mouse TNF ELISA kit (Invitrogen, 88–7324-88).
To measure TNF production, RAW264.7 cells were pretreated with 100 nM Wort, 20 nM Baf, 25 μM CQ, and 2 μM GW4869 for 2 h and then treated with 250 ng/mL LPS or 50 μM H2O2 for 24 h. The mouse TNF level was measured using a mouse TNF ELISA kit (Invitrogen, 88–7324-88).
4
Protein Expression and Cytokine Secretion
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Whole-cell lysates (30 μg protein/lane) were loaded in 10% SDS-PAGE gels and immunoblotted with Abs to p-AKT, Total-AKT, p-ERK, Total-ERK, p-IκBα, p52, Total β-catenin, Active β-catenin (Cell Signaling Technology), and RelA, RelB, p50, β-actin (Santa Cruz Biotechnology Inc.). Conditioned medium was collected from B cell culture and subjected to ELISA analysis. Mouse TNF ELISA kit (88-7324-22), mouse CCL3 ELISA kit (88-56013-22), human TNF ELISA kit (88-7346-22), and human CCL3 ELISA kit (88-7035-22), were purchased from Invitrogen and used according to the manufacturer’s instructions.
5
Cytokine Profiling in Skin Samples
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Cytokines were analyzed using a BioPlex Pro mouse cytokine 23-plex kit (Bio-Rad; Hercules, CA), or for analysis of TNF levels a mouse TNF ELISA kit (eBioscience; San Diego, CA) was used. Skin lysates were prepared by homogenizing skin in ice cold protein lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, 10% glycerol) using a Tissue Lyser II (QIAGEN; Hilden, Germany) for 12 cycles of 30 s at 30 Hz. A BCA kit (Thermo Fisher Scientific) was used to normalise protein levels. Values below the reference range were assigned the value of the lowest standard.
6
Quantifying TNF Secretion in Cells
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WT L929, Tnf KO L929, and Raw264.7 cells were grown in 12-well plate. WT and Tnf KO L929 cells were stimulated with 500 nM NCZ or 1 µM PTX in 300 µl fresh DMEM medium for 18 h; Raw264.7 cells were stimulated with 100 ng/ml LPS in 300 µl fresh DMEM medium for 3 h. Then, culture medium was collected and centrifuged at 8000 rpm for 2 min to remove cell debris. 50 µl aliquots were used to measure TNF concentration with mouse TNF ELISA Kit (eBioscience). Two independent wells were performed.
7
Ventral Hippocampal TNF Response to Stress
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Bilateral ventral hippocampal tissue was collected from stressed animals (24 h post forced swim stress) and their age-matched unstressed controls. They were anesthetized by isoflurane inhalation and decapitated for a rapid removal of the brain and gross dissection of the ventral hippocampi (<1 min). Tissue was then snap-frozen in liquid nitrogen. Tissue was then immediately homogenized using a handheld homogenizer for 5 s on ice in 400 µl of sterile PBS buffer containing a 1x protease inhibitor cocktail from Bioshop (PIC002.1). Samples were then centrifuged at 11,000 rpm for 20 min at 4 °C and the supernatants collected. TNF protein levels were measured according to the suppliers’ instructions of mouse TNF ELISA kit (eBioscience, Mouse TNF alpha ELISA Ready-SET-Go! Kit #88–7324). For standardization across samples, all TNF concentrations were divided by their corresponding crude-protein input concentrations as measured following the instructions of the bicinchoninic acid assay (BCA) kit from Thermo Fisher Scientific (#23227).
8
Quantifying Mouse TNF by ELISA
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The Mouse TNF ELISA kit by Thermofisher (Cat. # 88–7324) was used according to the manufacturer’s instructions.
9
Quantifying Mouse TNF by ELISA
10
Quantifying TNF in BV2 Cell Media
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The concentration of TNF in the BV2 cell conditioned media following treatment was measured using the ThermoFisher mouse TNF ELISA kit, a colorimetric sandwich ELISA, following the manufacturer's instructions. Values were interpolated from a standard curve of TNF and analyzed using Excel and GraphPad Prism software.
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