Ten milliliters of venous blood samples were collected into Vacutainer® EDTA tubes (Becton Dickinson, Madrid, Spain). Plasma fractions were obtained after two centrifugations, one at 1500× g for 10 min and the second at 2500× g for 15 min for the depletion of platelets, and stored at −80 °C until use. Cytokine analysis was performed using (i) Bio-Plex Pro™ Human Cytokine 27-plex Assay (Bio-Rad Laboratories Inc., CA, USA) for the analysis of the following cytokines: FGF-basic, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α and VEGF, following the instructions of the manufacturer. (ii) Human Luminex Discovery Assay (ref. LXSAHM, R&D Systems) for the analysis of IL-3, IL-16, IL-18, IL-23, IL-33 and CXCL1/GROα, and (iii) a Human TGF-β1 Elisa Kit (ref. RAB0460, from Sigma-Aldrich, Madrid, Spain) as a single ELISA kit for TGF-β1 detection, was used following strictly the instructions of the manufacturer. All determination analyses were performed using Luminex technology FLEXMAP 3D® equipment (Luminex Corporation, TX, USA), but for TGF-β1 colorimetric detection, a BioTek PowerWave HT microplate reader (BioTek Instruments, VT, USA) was used. All samples were measured in duplicate.
Human tgf β1 elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human TGF-β1 ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed to measure the concentration of human transforming growth factor beta 1 (TGF-β1) in cell culture supernates, serum, and plasma samples.
Automatically generated - may contain errors
Lab products found in correlation
Edta vacutainer tube, by BD (2 mentions)
Bio plex pro human cytokine 27 plex assay, by Bio-Rad (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Il 1α elisa kit, by R&D Systems (1 mentions)
Model 680, by Bio-Rad (1 mentions)
Human il 10 elisa kit, by R&D Systems (1 mentions)
Human tnf α elisa kit, by R&D Systems (1 mentions)
Lat a, by MedChemExpress (1 mentions)
11 protocols using human tgf β1 elisa kit
1
Plasma Cytokine Profiling Protocol
2
Quantifying TGF-β1 in Supernatant
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The TGF-β1 concentration in the supernatant was determined by a human TGF-β1 ELISA kit (R&D system, USA). BCA quantifying the total protein was conducted before ELISA and then the same protein quantity was loaded from different culture conditions. Absorbance was measured at a wavelength of 450 and 550 nm. The 450 nm values were subtracted by the 550 nm values for the correction of optical imperfections [40 (link)].
3
Immunofluorescence and ELISA Protocols
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Immunofluorescence assays were performed as previously described31 (link) and the images were captured with a fluorescence microscope (Leica).
ELISA was performed using a human TGF-β1 ELISA kit (R&D Systems) according to the manufacturer’s instructions.
ELISA was performed using a human TGF-β1 ELISA kit (R&D Systems) according to the manufacturer’s instructions.
4
TGF-β1 Quantification Protocol
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The TGF-β1 medium concentration was examined with the Human TGF-β1 ELISA kit (R&D System) following kit protocol.
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The TGF-β1 medium concentration was examined with the Human TGF-β1 ELISA kit (R&D System) following kit protocol.
5
KCOT, Ameloblastoma, and Follicular Cyst TGF-β1 and IL-1α Quantification
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Concentrations of TGF-β1 and IL-1α in KCOT, ameloblastoma, and follicular cyst intracellular fluid were determined using a human TGF-β1 ELISA kit and IL-1α ELISA kit (both from R&D Systems) while simultaneously measuring absorbance at 450 nm with a microplate reader (Model 680, Bio-Rad) in accordance with the manufacturer's instructions. Experimental results are presented as means ± standard deviation.
6
Quantification of Serum and PF TGF-β1
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The concentrations of serum and PF bioactive TGF-β1 in individual subjects were determined by ELISA using a human TGF-β1 ELISA Kit, according to the manufacturers’ instruction (R&D Systems, Minneapolis, MN, USA). Briefly, individual sera at 1:4 dilutions were subjected to ELISA analysis in triplicate, and the concentrations of serum and PF TGF-β1 in individual samples were calculated, according to the standard curve established using the recombinant TGF-β1 provided. The limitation of detection for TGF-β1 is 15.4 pg/ml.
7
Plasma TGF-β1 Quantification Protocol
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For each enrolled subject (Table S1 ), 5 mL of peripheral whole blood was collected using EDTA Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Plasma was obtained by centrifuging the whole blood at 1500 g for 15 min at 4 °C. Collected plasma samples were spun at max speed for 10 min at 4 °C and the supernatants were used for the assay. Hemolyzed samples were discarded. TGF-β1 plasma levels were detected by an ELISA kit (Human TGF-β1elisa kit, R&D Systems, Inc. Minneapolis, MN, USA) following the manufacturer’s instructions.
8
Quantifying TGF-β1 in Cell Media
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The amount of TGF-β1 in PMC culture medium was measured by using a human TGF-β1 ELISA kit from R&D Systems. The procedures were performed according to the manufacturer's instruction.
9
TGF-β1 Protein Quantification via ELISA
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The TGF-β1 concentration in the supernatant was determined by a human TGF-β1 ELISA kit (R&D Systems). BCA quantifying the total protein was conducted before ELISA; then the same protein quantity was loaded from different culture conditions. Absorbance was measured at a wavelength of 450 and 550 nm. The 450 nm values were subtracted from the 570 nm values for correction of the optical imperfections [39 (link)].
10
Macrophage polarization by HNSCC and BMSC sEVs
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Macrophages (1 × 106 cells/well) were seeded into six‐well plates and cocultured with 50 μg sEVs derived from HN6, HN6NT5EKO, SCC25, SCC25NT5EKO, BMSC, and BMSCNT5EOE. The experiments were divided into seven groups: M0, M2, M2+sEVsHNSCC, M2+sEVsHNSCC‐NT5EKO, M2+sEVsBMSC, M2+sEVsBMSC‐NT5EOE, and M2+sEVsBMSC‐NT5EOE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA). The CM was composed of RPMI1640 medium without FBS and was collected after incubation for 24 h. After centrifugation at 10,000×g for 10 min at 4°C, the coculture supernatant was added to a 96‐well plate to detect human IL‐6, IL‐10, TNF‐α, and TGF‐β1 using a human IL‐6 enzyme‐linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA), human IL‐10 ELISA kit (R&D Systems), human TNF‐α ELISA kit (R&D Systems), and human TGF‐β1 ELISA kit (R&D Systems) according to the manufacturer's instructions.
The procedure for extracting sEVs from serum samples has been previously described in 2.6. A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.
The procedure for extracting sEVs from serum samples has been previously described in 2.6. A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.
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