To characterize the paracrine activity of ASCs, conditioned medium was collected, centrifuged at 2,500 g to remove cell debris, and stored at –70 °C until the measurements were taken. To detect secreted proteins, conditioned medium was analyzed using the Proteome Profiler Human Angiogenesis Array Kit (R&D, USA) and Proteome Profiler Human Protease Array Kit (R&D, USA), according to the manufacturer’s instructions. The data were analyzed using Image Lab™ Software Version 5.0 (Bio-Rad, USA). VEGF-α, TGF-β, IL-6, IL-8, MCP-1, and IGF-1 concentrations in ASC conditioned medium were evaluated using the Human VEGF ELISA Set (Peprotech, USA), Human TGF-β1 DuoSet ELISA (R&D, USA), Human IL-6 ELISA Set (BD, USA), Human IL-8 ELISA Set (BD, USA), Human CCL2/MCP-1 DuoSet (R&D, USA), and Human IGF-1 DuoSet (R&D, USA), according to the manufacturer’s instructions.
Human tgf β1 duoset elisa
Manufactured by R&D Systems
Sourced in United States
The Human TGF-β1 DuoSet ELISA is a laboratory equipment product. It is an Enzyme-Linked Immunosorbent Assay (ELISA) kit used for the quantitative determination of human Transforming Growth Factor beta 1 (TGF-β1) levels in cell culture supernates, serum, and plasma samples.
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Lab products found in correlation
Image lab software version 5, by Bio-Rad (1 mentions)
Proteome profiler human angiogenesis array kit, by R&D Systems (1 mentions)
Human il 6 elisa set, by BD (1 mentions)
Human ccl2 mcp 1 duoset, by R&D Systems (1 mentions)
Proteome profiler human protease array kit, by R&D Systems (1 mentions)
Human igf 1 duoset, by R&D Systems (1 mentions)
Human il 8 elisa set, by BD (1 mentions)
Lipopolysaccharide binding protein, by Hycult Biotech (1 mentions)
Scd14, by R&D Systems (1 mentions)
I fabp, by R&D Systems (1 mentions)
Human cxcl10 ip 10 duoset elisa, by R&D Systems (1 mentions)
Human il 10 duoset elisa, by R&D Systems (1 mentions)
Duoset elisa ancillary reagent kit 1, by R&D Systems (1 mentions)
Human il 1β il 1f2 duoset elisa, by R&D Systems (1 mentions)
Human il 6 duoset, by R&D Systems (1 mentions)
Human il 6 elisaready set go kit, by Thermo Fisher Scientific (1 mentions)
Mouse tgf β1 duoset elisa, by R&D Systems (1 mentions)
Human il 1β elisa ready set go kit, by Thermo Fisher Scientific (1 mentions)
Anti hsp90 antibody, by Abcam (1 mentions)
8 protocols using human tgf β1 duoset elisa
1
Characterization of Paracrine Factors from Adipose Stem Cells
2
Biomarkers of Intestinal Injury and Inflammation
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CCR2 and CCR5 ligands (CCL2, CCL3, CCL4, CCL5) and proinflammatory cytokines (IL‐6, TNF‐α, IL‐1β, TGF‐β) were measured on the Luminex platform on plasma samples from Day 1 and during cenicriviroc treatment at Days 7 and 14 (HCYTOMAG‐60K MILLIPLEX (Merck Millipore, Darmstadt, Germany) was used for CCL2, CCL3, CCL4, IL‐1β, IL‐6; Human RANTES enzyme‐linked immunosorbent assay (ELISA) kit (PeproTech, London, UK) for CCL5; Human TGF‐β 1 DuoSet ELISA (R&D Systems, Minneapolis, MN) for TGF‐β). Markers of enterocyte death, intestinal fatty acid binding protein (I‐FABP) (R&D Systems), and bacterial translocation, flagellin (MyBioSource, San Diego, CA), lipopolysaccharide binding protein (LBP) (Hycult Biotech, Plymouth Meeting, Philadelphia, PA), and sCD14 (R&D Systems), were measured at the same timepoints by ELISA according to the manufacturers’ instructions. Correlation between flagellin and I‐FABP or ALT levels was evaluated in both cohorts.
3
Cytokine Quantification in pDC Cultures
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After 72-hours culture, MCL supernatants were collected and spin at 10000xg for 5 minutes to remove cell debris. IL-10, TGF-β1, and TGF-β2 concentration were determined by using the Human IL-10 DuoSet® ELISA (R&D Systems, cat. No. DY217B), Human TGF-β1 DuoSet® ELISA (R&D Systems; cat. No. DY240), and Human TGF-β2 DuoSet® ELISA (R&D Systems; cat. No. DY302), respectively, following the manufacturer’s instructions.
At 24 hours post-stimulation (as described in the Isolation, culture, and stimulation of human peripheral blood pDCs section), pDC supernatants were collected, as described above. The amounts of secreted IFN-α and CXCL10 were determined by using the Human IFN-α Matched Antibody Pair (eBioscience; cat. No. BMS216MST) and Human CXCL10/IP-10 DuoSet® ELISA (R&D Systems; cat. No. DY266). The supernatants were stored at -20°C until use.
At 24 hours post-stimulation (as described in the Isolation, culture, and stimulation of human peripheral blood pDCs section), pDC supernatants were collected, as described above. The amounts of secreted IFN-α and CXCL10 were determined by using the Human IFN-α Matched Antibody Pair (eBioscience; cat. No. BMS216MST) and Human CXCL10/IP-10 DuoSet® ELISA (R&D Systems; cat. No. DY266). The supernatants were stored at -20°C until use.
4
Quantitation of TGF-β and HIV p24
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TGF-β and HIVp24 proteins were quantitated by using ELISA. TGF-β levels and HIVp24 levels from harvested supernatants were determined using Human TGF-β 1 DuoSet ELISA (Cat # DY240, R&D Systems, Minneapolis, MN, USA) and HIV-1 p24 Antigen Capture Assay (Cat # 5447, ABL. Inc., Rockville, MD, USA), respectively, according to the manufacturer’s instructions.
5
TGF-β1 ELISA with Latent Activation
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ELISA was performed using the cell culture supernatant collected at 48 h after siRNA transfection to minimize variation due to the increased number of dead cells in the siRNA-treated samples. We collected the cell culture supernatant and centrifuged at 400× g for 5 min at 4 °C to remove the floating cells. We harvested the supernatant and centrifuged again at 10,000× g for 10 min at 4 °C to remove the debris. The supernatant was frozen at −80 °C until the analysis. TGF-β1 ELISA with the activation of latent TGF-β1 was performed using Human TGF-β1 DuoSet ELISA (R&D Systems DY240-05) with DuoSet ELISA Ancillary Reagent Kit 1 (R&D Systems DY007) according to the protocol recommended by the manufacturer. We subtracted the TGF-β1 level with the level of medium from no-cell control wells, treated with mock transfection. Then, the level of TGF-β1 was corrected with the number of living cells and normalized to siLuc.
6
Quantifying Cytokine Levels in Ascites
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The TNF-α, IL-1β, IL-6, and TGF-β1 concentrations of patient-derived ascites were assessed via enzyme-linked immunosorbent assay (ELISA) using the commercially available human TNF-α DuoSet® ELISA (Ref. DY210, R&D Systems, Switzerland), human IL-1β/IL-1F2 DuoSet® ELISA (Ref. DY201, R&D Systems, Switzerland), human IL-6 DuoSet® (Ref. DY206, R&D Systems, Switzerland), and human TGF-β1 DuoSet® ELISA (Ref. DY240, R&D Systems, Switzerland). So that the readings were within the detection limit, samples were diluted in reagent diluent. The following dilutions were used for the tested cytokines: undiluted for TNF-α and IL-1β, 1:8 (OvC3 and OvC4), and 1:40 (PeC) dilution for IL-6, and 1:10 for TGF-β1. Assays were performed according to the manufacturer’s instructions and measurements were conducted in triplicate.
7
Quantifying Cytokines in Extracellular Vesicles
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Cytokines were measured in EVs isolated from 5 × 105 human monocytes, 5 × 105 human M1 macrophages, 1 × 106 mouse monocytes, or 1 × 107 human/mouse whole-blood cells. TGF-β1 was measured with Human TGF-β1 DuoSet ELISA (R&D Systems, cat no. DY240) and mouse TGF-β1 DuoSet ELISA (R&D Systems, cat no. DY1679) or Human/Mouse TGF-β1 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-8350-88). IL-10, IL-6, and Il-1β were measured using the human IL-10 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7106-22), human IL-6 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7066-22), or human IL-1β ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7261-86). In all cases, the ELISA was performed according to the manufacturer’s protocol. CD9 and HSP90 were measured with ELISA on immobilized vesicle using anti-CD9 antibody (Novus Biologicals, cat no. NB500-327) (2 µg/mL) and anti-HSP90 antibody (Abcam, cat no ab79848) (2 µg/mL), respectively. Vesicles in different fractions of size-exclusion chromatography was immobilized with TGF-β1 capture antibody from Human TGF-β1 DuoSet ELISA, and CD9 or HSP90 was detected on vesicles by biotinylated CD9 and HSP90 antibody and streptavidin-conjugated HRP.
8
Quantification of Total and Active TGF-β1
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Both the amount of total TGF-β1 and active TGF-β1 in collected culture media was quantified using the human TGF-β1 DuoSet ELISA (R&D systems). To measure the total amount of TGF-β1 in each sample, an acidic activation step was performed as per the manufacturer's instructions. Analyses of the samples without this activation step provided the amount of active TGF-β1 within the sample.
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