Levels of IL-10, IL-35 and TGF-β were determined in supernatants from Treg cultures with the Quantikine ELISA Mouse IL-10 Immunoassay (R&D Systems), the LEGEND MAX Mouse IL-35 Heterodimer ELISA Kit or the LEGEND MAX Total TGF-β1 ELISA Kit (BioLegend). IL-2 levels in supernatants of differentiating Th17 cells were measured with the BD Cytometric Bead Array, Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences). All steps were performed according to the manufacturers’ instructions.
Legend max total tgf β1 elisa kit
Manufactured by BioLegend
Sourced in United States
The LEGEND MAX Total TGF-β1 ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of total transforming growth factor beta 1 (TGF-β1) in cell culture supernatants, serum, plasma, and other biological fluids.
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Lab products found in correlation
Legend max mouse il 35 heterodimer elisa kit, by BioLegend (2 mentions)
Cytometric bead array mouse th1 th2 th17 cytokine kit, by BD (1 mentions)
Simplestep elisa kit, by Abcam (1 mentions)
Magnetic luminex assay, by R&D Systems (1 mentions)
Dasit xt 1800j, by Sysmex (1 mentions)
Human cytokine magnetic 30 plex panel, by Thermo Fisher Scientific (1 mentions)
Gen5 microplate reader and imager software, by Agilent Technologies (1 mentions)
Legend max free active tgf β1 elisa kit, by BioLegend (1 mentions)
Synergy lx, by Agilent Technologies (1 mentions)
Elisa max deluxe, by BioLegend (1 mentions)
Elisa max standard set, by BioLegend (1 mentions)
Pre coated plates, by BioLegend (1 mentions)
Multiskan ex microplate reader, by Thermo Fisher Scientific (1 mentions)
Il 10 mouse elisa kit, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
10 protocols using legend max total tgf β1 elisa kit
1
Quantifying Treg and Th17 Cytokines
2
Quantifying Cytokine Secretion in Immune-Tumor Interactions
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For IFN-γ secretion measurement, human purified NK cells were stimulated with IL-2 (100 IU/ml). After 72 h, activated NK cells were washed twice and mixed with untreated or SR141716-treated glioma target cells in 200 μl of complete medium. Cells were incubated for the indicated time. Thereafter, supernatants were collected and stored at −20°C pending measurement. The concentration of IFN-γ was measured by ELISA assay according to manufacturer's specification (R&D Systems, Minneapolis, MN). For TGF-beta1 secretion measurement, human primary glioma cell lines were plated into p60 tissue culture plates at a densities of 20 × 103 cells/cm2 and were allowed to grow for 24 h. Afterwards cells were washed with PBS and treated with vehicle or test substance in complete medium. After 24 h supernatants were collected and stored at −20°C pending measurement. The concentration of TGF-beta1 was measured by ELISA assay (LEGEND MAX™ Total TGF-β1 ELISA Kit, BioLegend, San Diego, CA, USA). Cells were treated in triplicates and ELISA were done in duplicates for each cell sample.
3
TGF-β1 Quantification in Cell Culture
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Cells were grown in plates for experiments. After treatment, culture medium of cells was collected to measure TGFβ1 concentrations with TGFβ1 Emax® ImmunoAssay Systems (Promega) for HK-2 cells or LEGEND MAX™ Total TGF-β1 ELISA kit (BioLegend, San Diego, CA) for RTECs.
4
Cytokine and Chemokine Analysis of BALF
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At 7, 14, and 21 days, after the in vivo imaging, subsets of 5 (Sal), 5 (BLM), 5 (Sal + ICG), and 12 (BLM + ICG) mice were euthanized with an overdose of anesthetic followed by bleeding from the abdominal aorta. Bronchoalveolar lavage fluid (BALF) was then harvested by gently washing the lungs with 0.6-mL sterile Hank’s balanced salt solution (HBSS) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 100 mM 4-(2-hydroxy-ethyl)-1-piperazineethansulphonic acid (HEPES); the collected fluid was centrifuged at 300 g for 10 min at 4°C. The cell pellet was resuspended in 0.2 mL of phosphate buffer solution (PBS), and leukocytes and cell populations were determined using an automated cell counter (Dasit XT 1800 J, Sysmex), while supernatants were frozen at −80°C. At a later time, BAL supernatants were thawed, and the levels of multiple cytokines and chemokines were determined, using a Magnetic Luminex Assay (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions; neutrophil elastase and total TGF-β1 levels were determined with the SimpleStep ELISA Kit (Abcam) and the LEGEND MAX Total TGF-β1 ELISA Kit (Biolegend), respectively.
5
Quantification of Cytokine Secretion
6
TGF-β1 Activity Quantification by ELISA
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The supernatants were collected and centrifuged at 500g and 4°C for 5 minutes, and the supernatants were collected carefully and stored at –20°C prior to ELISA. TGF-β1 activity was estimated by ELISA using a LEGEND MAX Free Active TGFβ-1 ELISA Kit and LEGEND MAX Total TGF-β1 ELISA Kit (BioLegend) according to the manufacturer’s protocols. Briefly, standards and 50 μL of supernatants were loaded into the wells, and the absorbance were measured at 450 nm using a Synergy-LX multimode reader (BioTek) and analyzed by Gen5 Microplate Reader and Imager software (BioTek). Each of the standard and samples were tested in duplicate.
7
Quantification of Cytokine Levels in Melanoma and Macrophage Samples
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CM from melanoma cells or supernatants from macrophages were used for sandwich ELISA experiments using commercially available kits (see also the table for reagents above). 96-well NUNC clear flat-bottom plates were coated with antibodies (IL-10, IL-8, IL-1α and TNF-α) and incubated O/N at 4°C in a humified chamber. ELISA MAX™ standard sets (Biolegend) were used for the detection of IL-10, IL-8 and TNF-α; ELISA MAX Deluxe (Biolegend) for IL1-α; and for TGF-β, LEGEND MAX Total TGF-β1 ELISA Kit with pre-coated plates was used (Biolegend). Microwell absorbance was read at 450 nm on a Thermo Scientific Multiskan EX microplate reader. All samples were quantified based on a standard curve using Microsoft Excel.
8
Quantifying TGFβ, IL-10, and IL-35 in Ascites
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For the detection of transforming growth factor-β (TGFβ), IL-10 and IL-35, ascites was collected from tumor-bearing triple oncogene IP/EP mice by 18-gauge needles before they were sacrificed. For the double oncogene IP/EP and naïve mice, 3 mL of PBS was IP injected to the mice and mix gently for 1 min, followed by removal of the peritoneal washed fluid by 18-gauge needles. TGFβ levels were measured from the cell-free ascites using LEGEND MAX Total TGF-β1 ELISA Kit (Biolegend), IL-10 levels were measured by the Mouse IL-10 ELISA Kit (Thermo Fisher), and IL-35 levels were measured by the LEGEND MAX Mouse IL-35 Heterodimer ELISA Kit (Biolegend) following the manufacturers’ instructions.
9
Quantifying TGFβ1 in KG1a Supernatant
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KG1a were cultured in SF‐03(Sekisui medical) medium for 3 days. Then, the supernatant was collected and TGFβ1 concentration was measured with LEGEND MAX Total TGF‐β1 ELISA Kit (Biolegend) according to the manufacturer's instructions.
10
Measurement of Serum Total TGF-β1 by ELISA
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The concentrations of total TGF-β1 in serum were measured using the Legend Max Total TGF-β1 ELISA kit (BioLegend, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, the capture antibody-coated wells in Nunc C bottom immunoplates supplied in the kit were washed 3 times with washing solution. The serum samples and standards were then added to the wells, after which the plates were incubated at room temperature for 2 h. After washing 3 times, TGF-β1 detection antibody solution was added to each well followed by incubation at room temperature for 1 h with shaking. The wells were then washed with washing solution, after which HRP-conjugated detection antibodies were diluted 5,000-fold with conjugate diluent (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween-20, pH 8.0) and transferred to each well. The plates were subsequently incubated at room temperature for 30 min, then washed 3 times with washing solution. An enzyme reaction was initiated by adding substrate solution and incubating the plate at room temperature in the dark for 30 min. Finally, the reaction was terminated by adding a stop solution, and the absorbance at 450 nm was measured within 30 min using a microplate reader (Molecular Device, Sunnyvale, CA, USA).
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