BALs were collected at 18 hpi by flushing the lungs of euthanized and intubated mice with 0.5 mL PBS (3 times) containing an anti-protease cocktail (Complete from Roche Diagnostics) and 5 mM EDTA. The collected fluids were centrifuged and both the pellets and the supernatants were snap-frozen and stored at −80 °C. Protein concentration in the supernatants was measured using the BCA kit from Thermo Scientific. IL-6 levels were measured using the mouse IL-6 ELISA kit from BioLegend. To evaluate the hemoglobin contents, pellets were thawed, resuspended in water and centrifuged. The supernatants were read at OD560. Dosages were performed once in triplicate.
Mouse il 6 elisa kit
Manufactured by BioLegend
Sourced in United States, China
The Mouse IL-6 ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of mouse interleukin-6 (IL-6) in cell culture supernatants, serum, and plasma samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Mouse tnf α elisa kit, by Dakewe (2 mentions)
Human il 6 elisa kit, by BioLegend (1 mentions)
Mouse tnf elisa kit, by BioLegend (1 mentions)
Mouse tnf α elisa kit, by BioLegend (1 mentions)
Criterion xt bis tris gel, by Bio-Rad (1 mentions)
Xt mes buffer, by Bio-Rad (1 mentions)
Image lab analysis software, by Bio-Rad (1 mentions)
Phospho akt ser473 4060, by Cell Signaling Technology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Mouse il 1β elisa kit, by Thermo Fisher Scientific (1 mentions)
Model elx800, by Agilent Technologies (1 mentions)
11 protocols using mouse il 6 elisa kit
1
Bronchoalveolar Lavage Fluid Analysis
2
Multimodal Antibody Profiling of Stem Cells
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The following antibodies were used in this study: APC anti-human CD44v3 antibody (AB5088A, 1:300) was purchased from R&D Systems, (Minneapolis, MN). AF700 anti-mouse/human CD44 antibody (103025, 1:300), FITC anti-human CD81 antibody (cat. 349503, 1:300), APC anti-mouse/human CD44 Antibody (103012, 1:300), PE-Cy7 anti-human CD133 Antibody (393910, 1:300), and APC anti-mouse CD81 (104909, 1:300), and Zombie Aqua (423101), were purchased from Biolegend (San Diego, CA). Human FITC-Heparan Sulfate antibody (H1890-10, 1:300) was purchased from US Biological (Salem, MA). Human IL-6 ELISA kit and Mouse IL-6 ELISA kit were purchased from Biolegend (San Diego, CA).
Neutralizing anti-human IL-6 antibody (mabg-hil6-3) was purchased from In vivogen (San Diego, CA). Heparinase I/III blend (H3917-50UN), chondroitinase ABC (C2905-2UN), and JSI-124 (C4493-1MG) were purchased from Sigma-Aldrich (Saint Louis, MO). PDZ1i was generously provided by Dr. Paul B. Fisher (23 (link)).
Neutralizing anti-human IL-6 antibody (mabg-hil6-3) was purchased from In vivogen (San Diego, CA). Heparinase I/III blend (H3917-50UN), chondroitinase ABC (C2905-2UN), and JSI-124 (C4493-1MG) were purchased from Sigma-Aldrich (Saint Louis, MO). PDZ1i was generously provided by Dr. Paul B. Fisher (23 (link)).
3
Cytokine Quantification in Murine Colon
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TNF, IL1B, IL6, and CXCL1 in colon homogenates or serum were measured using mouse TNF ELISA kit, mouse IL6 ELISA kit, mouse IL1B ELISA kit (Biolegend, San Diego, CA, USA) and mouse CXCL1 ELISA Kit (Invitrogen, Carlsbad, CA, USA) accordingly to the manufacturer’s instruction.
4
Quantification of Inflammatory Mediators
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(Soluble) TNF-α, TGF-β1, IL-6, IL-6R and TNFR-1 in the medium of BV2 cells or in the supernatants of brain tissue homogenates (see Supplementary Materials ) were quantified using mouse TNF-α ELISA kit (430901, BioLegend, San Diego, CA, USA), human/mouse TGF-β ELISA kit (88-8350-88, Invitrogen, Waltham, MA, USA), mouse IL-6 ELISA kit (431301, BioLegend, USA), mouse IL-6R ELISA Kit (RAB0314, Sigma) and Mouse/Rat TNF R-1 Quantikine ELISA Kit (MRT10, R&D Systems), using manufacturer’s instructions. Due to the strong inhibition of constitutive IL-6 release from BV2 microglial cell by pCS (1, 5, 10 and 50 μM) treatment, IL-6 concentrations in the medium after exposure to pCS (1, 5,10 and 50 μM) were under the detection limit that was set as ‘0′.
5
Quantifying AMPK and AKT Phosphorylation
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Criterion XT Bis-Tris Gels (4–12% polyacrylamide, BioRad: Hercules, CA, USA), XT MES Buffer (BioRad) and Trans-Blot® Turbo™ Midi Transfer Packs were used for electrophoresis and immunoblotting (n = 2–4) on PVDF membranes. Phospho-AMPKα (Thr172; 2531) and Phospho-AKT (Ser473; 4060) antibodies were sourced from Cell Signaling Technology: Danvers, MA, USA. Images were obtained with a ChemiDoc + XRS and immunoblots were quantified with ImageLab analysis software (BioRad). IL-6 was quantified in the conditioned media using a commercially available mouse IL-6 ELISA kit (n = 10; 431301, BioLegend: San Diego, CA, USA).
6
Quantifying IL-6 in Schu S4-Infected Macrophages
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At 24 h p.i., culture supernatants from BMMs infected with the Schu S4 strains were collected following approved BSL3 guidelines. Levels of IL-6 were determined using a mouse IL-6 ELISA kit (Biolegend) according to the manufacturer’s protocol. Values were recorded as picograms per milliliter.
7
Evaluating DEX's Anti-inflammatory Activity
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The biological activity of DEX was evaluated in vitro using a test system involving the LPS treatment of DC2.4 dendritic cells. To validate the system, DC2.4 cells were first plated onto a 24-well tissue culture plate at 5 × 104 cells per well and cultured overnight with or without LPS at a concentration of 1 μg/ml. Then, supernatant was collected, and the concentration of IL-6 was measured using a mouse IL-6 ELISA kit (BioLegend) according to the manufacturer’s protocol. To compare free DEX and VLA-DEX-NP, the two formulations were first added to the culture medium at final drug concentrations of 0.01, 0.1, and 1 μM, followed by 2 hours of incubation. For free DEX, 1000× stock solutions were prepared at 0.01, 0.1, and 1 mM in dimethyl sulfoxide. Then, the cells were treated with LPS overnight before measuring the concentration of IL-6 in the supernatant. To test the effect of empty nanoparticles, either PLGA cores or VLA-NP at a final concentration of 1 μg/ml were first incubated with the cells for 2 hours, followed by an overnight incubation either with or without LPS before measuring IL-6 levels.
8
BALF Cytokine Measurement in RT-Treated Mice
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After RT treated, mice lungs were irrigated three times with 1.0 ml PBS. The collected bronchoalveolar lavage fluid (BALF) was immediately centrifuged to separate the cells and supernatant. Measurement of cytokine in the supernatants of BALF and RT-treated RAW264.7 cells was carried out using Mouse TNF-α ELISA kit (Dakewe, Shenzhen, China) and Mouse IL-6 ELISA kit (Biolegend, San Diego, CA, United States) according to the manufacturer’s instructions.
9
Mouse Cytokine Detection by ELISA
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Mouse IL-1β ELISA Kit was purchased from Invitrogen (#BMS6002, Thermo Fisher Scientific, MA, USA). Mouse IL-6 ELISA Kit was obtained from Biolegend (#431304, San Diego, CA, USA). The measurement was performed according to manufacturer’s protocols.
10
ELISA Assay for Colon IL-6 Quantification
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(ELISA) %ssay 2・8・1 IL-6 level Proteins were extracted from the frozen colon using T-PER Tissue Protein Extraction Reagent Kit according to the manufacturer's instruc tions. Protein concentrations were determined by BCA protein assay kit (Rockford, IL, USA) and equal amounts of protein were utilized in triplicate for each condition. The concentrations of IL-6 in the colons were mea sured using a mouse IL-6 ELISA kit (Biolegend) ac cording to the manufacturers' instructions. All samples were assayed in triplicate. Absorbance was read at 450 nm using a microplate reader (model ELX800; BioTek Instruments, Inc., Winooski, VT, USA). Results are presented as mean (expressed in pg/mg total pro tein) and standard deviations ("#).
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