For the analysis of biochemical markers, 5 ml of the venous blood sample was collected under aseptic conditions by venipuncture and divided into three parts. From the first part (2 ml), the blood was allowed to clot, and the serum was separated. From the second part (2 ml), plasma was isolated by centrifugation at 2000 × g for 15 min at 4°C. RNA was isolated from the remaining third part (1 ml) of the blood. The serum and plasma were stored at −80°C until analyzed. ELISA kits used for the estimation of levels of IL-6 (Gen-Probe, Diaclone Diagnostic, France), IL-17A (Gen-Asia Biotech, China), TNF-α (Gen-Probe, Diaclone Diagnostic, France), BDNF (Raybiotech, Inc), DHEAS (Qayee Bio-Technology), β-endorphin (Phoenix Pharmaceuticals, Inc.) and sirtuin 1 (Qayee Bio-Technology). Serum TGF-β levels were estimated by a magnetic bead-based multiplex assay using Bio-Plex Pro TGF-β Assays (Bio-Rad Laboratories Inc., United States) according to manufacturer’s guidelines.
Bio plex pro tgf β assay
Manufactured by Bio-Rad
Sourced in United States
The Bio-Plex Pro TGF-β assay is a multiplex immunoassay designed to quantify transforming growth factor beta (TGF-β) levels in a variety of sample types. The assay utilizes magnetic beads coated with specific antibodies to capture and detect TGF-β proteins. This technology allows for the simultaneous measurement of multiple TGF-β isoforms in a single well.
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Lab products found in correlation
Aia system, by Tosoh (3 mentions)
Bio plex pro mouse cytokine 23 plex, by Bio-Rad (2 mentions)
Ifn betamouse procartaplex simplex kit, by Thermo Fisher Scientific (2 mentions)
Il 28b ifn lambda 3 mouse elisa kit, by Thermo Fisher Scientific (2 mentions)
T per, by Thermo Fisher Scientific (2 mentions)
Bio plex 200, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Bio plex manager software, by Bio-Rad (2 mentions)
Bio plex pro human cytokine 21 and 27 plex immunoassays, by Bio-Rad (2 mentions)
Brain derived neurotrophic factor (bdnf), by RayBiotech (1 mentions)
β endorphin, by Phoenix Pharmaceuticals (1 mentions)
Synergy htx multi mode reader, by Agilent Technologies (1 mentions)
Luminex 200 analyzer, by Bio-Rad (1 mentions)
Luminex assay kit, by R&D Systems (1 mentions)
Quantikine human coagulation factor 3 tissue factor immunoassay, by R&D Systems (1 mentions)
Human serpin e1 pai 1 quantikine elisa kit, by R&D Systems (1 mentions)
12 protocols using bio plex pro tgf β assay
1
Biomarker Quantification Protocol
2
Quantifying Soluble TGF-β Isoforms
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The soluble TGF-β (sTGF-β) isoforms were quanti ed in serum by a multiplex test based on magnetic beads "Bio-Plex Pro™ TGF-β Assays" (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer's instructions. All samples and controls were analyzed in duplicate; the coe cients of variation were within the acceptable range (<20%). The assay's sensitivity was 6.35 pg/mL for sTGF-β1, 4.57 pg/mL for sTGF-β2, and 4.12 pg/mL for sTGF-β3 (data were obtained from the results issued by the Bio-Plex Magpix instrument). Data were tted to a ve-parameter logistic function.
3
Cytokine Profiling of Lung Tissue after Vaccine Adjuvants
4
Cytokine Analysis in Lung Tissue
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At 24 and 48 hours after vaccination with OVA and various adjuvants, lungs were harvested for cytokine analysis Five hundred microliter of the mammalian tissue protein extraction reagent (T-PER) (Thermo Scientific) containing protease inhibitor cocktails (Roche, Indianapolis, IN), was added to the lung tissue and homogenized in a tissue homogenizer. Subsequently, tissue lystate was centrifuged at 10,000 x g for 5 minutes at 4C. Supernatants were collected and protein concentrations in extracts were quantified using BCA method and 1mg of total lysate was added for each well in a 96-well plate. Cytokines were quantified using Bio-Plex Pro Mouse Cytokine 23-plex and Bio-Plex Pro TGF-β Assays (Bio-Rad), IFN beta Mouse ProcartaPlex Simplex Kit and IL-28B/(IFN lambda 3) Mouse ELISA kit (eBioscience), as recommended by the manufacturer. The samples were acquired and analyzed using Bioplex-200 with the Bioplex Manager 6.1.1. The data were normalized by (the amount of cytokine/ml) ∗ (extraction volume) divided by the weight of the lung tissue (mg).
5
Measuring TGF-β Isoforms and Activity
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TGF-β isoforms were measured with Bio-Plex Pro TGF-β Assays (Bio-Rad) in accordance with the manufacturer’s directions. To measure TGF-β activity, the NIH/3T3 SMAD2/3-luciferase reporter cell line (Signosis) was cocultured with BALF and extracted with RPMI 1640 (3:1 v/v ratio) or platelet-free plasma at a 1:10 (v/v) ratio for 16 hours. Luciferase activity was measured using a BioTek Synergy/HTX Multi-Mode Reader.
6
Measuring Aqueous Humor Biomarkers
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The level of ATX in the AH was determined using a two-site immunoenzymatic assay with an ATX assay reagent equipped with a Tosoh AIA system (Tosoh, Tokyo, Japan) as described previously27 (link),28 (link),54 (link),55 (link). The TGF-β levels in the AH were measured using the Bio-Plex Pro TGF-β Assay (Bio-Rad, CA, USA) following the manufacturer’s protocol.
7
Multiplex Fibrosis Biomarker Quantification
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The secreted fibrosis markers were measured in the serum samples of patients by multiplex fluorescent-bead-based technology (Luminex 200, Austin, TX, USA) using two commercial Luminex screening assay kits: a customized Luminex Assay kit from R&D Systems (Lilles, France) for MMP-1, resistin, Collagen IA1, PDGF-BB and TIMP-1 and the Bio-Plex Pro TGF-β assay from Bio–Rad Laboratories (Marnes-la-Coquette, France).
In brief, the samples were diluted before incubation with specific antibody-coated fluorescent beads according to the manufacturer’s recommendations. After washing, 50 beads were analyzed with the Luminex 200™ analyzer and Bio-Plex Manager software version 6 (Bio–Rad Laboratories), and the analyte concentrations of the samples were estimated through the serial dilution of cytokine standards (MMP-1 sensitivity < 3 pg/mL; resistin sensitivity < 20 ng/mL; Col IA1 sensitivity < 100 pg/mL; PDGF-BB sensitivity < 50 pg/mL; TIMP-1 sensitivity < 800 pg/mL; and TGF-β1 sensitivity < 15 pg/mL).
In brief, the samples were diluted before incubation with specific antibody-coated fluorescent beads according to the manufacturer’s recommendations. After washing, 50 beads were analyzed with the Luminex 200™ analyzer and Bio-Plex Manager software version 6 (Bio–Rad Laboratories), and the analyte concentrations of the samples were estimated through the serial dilution of cytokine standards (MMP-1 sensitivity < 3 pg/mL; resistin sensitivity < 20 ng/mL; Col IA1 sensitivity < 100 pg/mL; PDGF-BB sensitivity < 50 pg/mL; TIMP-1 sensitivity < 800 pg/mL; and TGF-β1 sensitivity < 15 pg/mL).
8
Biochemical Assays for Ocular Biomarkers
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The level of ATX in AH was determined using a two-site immunoenzymetric assay with an ATX assay reagent by means of a Tosoh AIA system (Tosoh, Tokyo, Japan), as described previously [11 (link), 12 (link), 18 (link)]. TGF-β levels in AH were measured using the Bio-Plex Pro TGF-β Assay (Bio-Rad, Hercules, CA, USA), in accordance with the manufacturer’s protocol. LysoPLD activity in the culture medium was determined as previously described [11 (link), 12 (link), 18 (link)].
9
Cytokine and Angiogenic Factor Profiling
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Plasma samples were analyzed for 51 plasma cytokines and angiogenic factors: TGF-β1, TGF-β2, TGF-β3, IFN-α2, IL-1α, IL-2Rα, IL-3, IL-12p40, IL-16, IL-18, CTACK, Gro-α, HGF, LIF, MCP-3, M-CSF, MIF, MIG, β-NGF, SCF, SCGF-β, SDF-1α, TNF-β, TRAIL, IL-1β, Il-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF basic, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF bb, RANTES, TNF-α, VEGF using predesigned panels as described previously and were available for subset of patients (Bio-Plex Pro TGF-β assay, Bio-Plex Pro Human Cytokine 21- and 27-plex immunoassays; Bio-Rad Laboratories, Hercules, CA, USA) [22 (link)]. The large panel of cytokines was analyzed as data were available from the previous study [22 (link)].
10
Aqueous Humor Assay for ATX and TGF-β
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AH samples were collected as described previously4 (link),10 (link). Levels of ATX in the AH were determined via a two-site immunoenzymatic assay with an ATX assay reagent using a Tosoh AIA system (Tosoh, Tokyo, Japan). TGF-β levels in the AH were measured using a Bio-Plex Pro TGF-β assay (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer’s protocol.
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