Confluent monolayers of cells were incubated in serum-free medium for 48 h to generate conditioned medium. The conditioned medium was then transferred to fresh plates of MRC5 cells for 24 h or analyzed for active TGF-β levels using a commercial ELISA (R&D Quantikine ELISA Assay). In some experiments SB431452 (10 µM) was added to the conditioned medium. After 24 h the MRC5 cells were appropriately lysed for experiments as described.
Quantikine elisa assay
Manufactured by R&D Systems
Sourced in United States
Quantikine ELISA assays are quantitative sandwich enzyme-linked immunosorbent assays designed for the measurement of specific proteins in cell culture supernates, serum, plasma, and other biological fluids.
Automatically generated - may contain errors
Lab products found in correlation
Duoset elisa, by R&D Systems (2 mentions)
Oxldl, by Immundiagnostik (1 mentions)
Platinum elisa kit, by Thermo Fisher Scientific (1 mentions)
Cobas 8000, by Roche (1 mentions)
Vacutainer, by BD (1 mentions)
Cck 8, by R&D Systems (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Luminex xmap platform, by Merck Group (1 mentions)
Mep00b, by R&D Systems (1 mentions)
Micro hematocrit centrifuge, by Unico (1 mentions)
Opsys mr, by Dynex (1 mentions)
17 protocols using quantikine elisa assay
1
Conditioned Medium Activates TGF-β Signaling
2
Plasma Biomarker Profiling in Clinical Studies
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Participants fasted overnight and took their morning medication(s) after the study visit. Their last meal was not standardised. Fasting blood samples were obtained and the residual supernatant plasma stored at −80°C prior to batch analysis. HbA1c, glucose, liver, kidney function and lipid profile were analysed according to standard operating procedures in the accredited laboratory at University Hospitals Leicester NHS Trust.
Insulin and leptin were quantified by multiplex assay on a Luminex platform, as previously described.8 Plasma Adiponectin was analysed using a Quantikine ELISA assay (R&D Systems, Minneapolis, USA). All samples were assayed in duplicate (the mean reported), with all samples having a co‐efficient of variance (%CV) ≤ 20%.
The Luminex multiplexed plasma biomarker assay (Bristol Myers Squibb, Ewing Township, New Jersey, USA) quantifies multiple biomarkers in a single plasma sample. Each sample was analysed in duplicate with the mean reported and repeated where the CV exceeded 30%. The panel includes markers reflecting inflammation, fibrosis, myocardial injury, reactive oxygen species. Luminex® Assays and Luminex High Performance Assays: R&D Systems. 2017 (https://www.rndsystems.com/products/luminex‐assays‐and‐high‐performance‐assays ).The full list of biomarkers of interest is provided in supplemental material 1.3.
Insulin and leptin were quantified by multiplex assay on a Luminex platform, as previously described.
The Luminex multiplexed plasma biomarker assay (Bristol Myers Squibb, Ewing Township, New Jersey, USA) quantifies multiple biomarkers in a single plasma sample. Each sample was analysed in duplicate with the mean reported and repeated where the CV exceeded 30%. The panel includes markers reflecting inflammation, fibrosis, myocardial injury, reactive oxygen species. Luminex® Assays and Luminex High Performance Assays: R&D Systems. 2017 (
3
Evaluating BCA101 Serum Levels in Mice
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Single dose of BCA101 (5 mg/kg) was injected intravenously to 5–6 weeks old female BALB/c mice (n = 3) and mice were bled through retro-orbital plexus at regular intervals. Serum samples were diluted in assay diluent (0.1% PBST plus blocking buffer) and bifunctional ELISA was performed as described above. For TGFβ serum levels, samples were treated with 1N HCl for 10 minutes and neutralized with 1.2N NaOH/0.5 mol/L HEPES buffer. Activated TGFβ levels were measured by quantikine ELISA assay as per the manufacturer's instructions (R&D Systems).
4
Substance Use and Gut Microbiome in HIV
5
Biomarkers for Kidney Function Assessment
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N-Terminal - brain natriuretic peptide (NT-BNP) was measured using the Cobas 6000 601e assay (Roche, Mississauga, Ontario, Canada). We also measured the growth differentiation factor-15 (GDF-15), a novel biomarker expressed in response to tissue injury with elevations implicated in worsening kidney function among patients with CKD [22 (link)], using Quantikine ELISA assay (R&D Systems Inc., Minneapolis, Minnesota, USA).
6
Quantitative VEGF Secretion in hMSC Cultures
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A Quantikine® ELISA assay (R&D Systems) was used to detect VEGF
level in cell culture supernatant. Cell culture supernatant was harvested at day
1 and 7 from hMSCs cultured on CoO 0%, 2% and Synthemax II microspheres in low
attachment 96 well plates. Cell culture supernatant was collected from two
separate experiments, performed using two different donors. Samples of cell
culture supernatant were centrifuged at 13,000 rpm for 20 min at 4°C and stored
at −80°until processing. The ELISA assay was performed according to manufacturer
instructions and normalised per DNA content, as quantified by
Picogreen®.
level in cell culture supernatant. Cell culture supernatant was harvested at day
1 and 7 from hMSCs cultured on CoO 0%, 2% and Synthemax II microspheres in low
attachment 96 well plates. Cell culture supernatant was collected from two
separate experiments, performed using two different donors. Samples of cell
culture supernatant were centrifuged at 13,000 rpm for 20 min at 4°C and stored
at −80°until processing. The ELISA assay was performed according to manufacturer
instructions and normalised per DNA content, as quantified by
Picogreen®.
7
Quantifying Inflammatory Markers in AMD
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The concentration of IL-6, IL-8 and VEGF in the cell culture medium was measured using the Quantikine ELISA assay (R&D Systems). The concentration of PTX3 both in the conditioned medium of ARPE-19 cells and in the vitreous humor of AMD and non-AMD donors was assessed using an in-house developed ELISA assay (Canovi et al., 2014 (link)).
8
Quantifying VEGF Secretion in MG-63 Cells
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A human Quantikine ® ELISA assay (R&D Systems, Bio-Techne, Abingdon, UK) was used to detect VEGF levels in cell culture supernatant. A monolayer of phosphate glass microspheres (30mg/well) was placed into low-adhesion 24-well plates (Appleton Woods, UK) and then UV sterilized. MG-63 cells were seeded on the microspheres at a concentration of 9 x 10 4 cells per well and incubated at 37°C/5%CO 2 . Cells grown on standard tissue culture 24-well plates were used as a control. Media was replaced every two days and cell culture supernatant was harvested at 24 and 72hrs post-seeding. Samples of cell culture supernatant were centrifuged at 13,000rpm for 20mins at 4°C and stored at -80°C until processing. The ELISA assay was performed according to manufacturer instructions. The assay was repeated two times and each condition was analysed in duplicates. Data are presented as mean ± standard variation (n=4).
9
Plasma Biomarkers Measurement Workflow
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According to each manufacturer's specifications, IL-6 and TNF-α plasma levels were determined by using the high sensitivity Quantikine ELISA assays (R&D systems, Minneapolis, Minnesota); the Platinum ELISA kit from eBioscience, Ltd. (Hatfield, Ireland, United Kingdom) was employed for IL-10; oxLDL levels were determined by using ox-LDL Immundiagnostik AG - Bensheim, Germany ELISA Kit.
10
Biomarker Assessment of Cardiovascular Risk
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Venous blood was drawn from the brachial vein in a fasted state, between 08:00 and 10:00 h, in 10 ml EDTA tubes (Vacutainer, BD; Franklin Lakes, NJ, USA). Within 3 h, tubes were centrifuged (Hettich Rotina 420R, radius 183 mm; 3800 rpm (2954 g ), 10 min, room temperature), and plasma was collected and stored at −80°C until assayed. Plasma IGF1 levels were determined by a chemiluminescent immunometric assay (Liaison, DiaSorin, Saluggia, Italy) according to the 1st WHO International Standard for insulin-like growth factor 1 (NIBSC code: 02/25). Levels of total cholesterol, LDL cholesterol, HDL cholesterol, non-HDL cholesterol and triglycerides were determined using an in-house analyzer (Cobas 8000; Roche Diagnostics).
Plasma E-selectin, matrix metalloproteinase (MMP)2, vascular cell adhesion molecule (VCAM)1, high-sensitivity C-reactive protein (hsCRP), interleukin (IL)18, IL18-binding protein (IL18BP) and IL6 levels were determined with ELISA. For E-Selectin, MMP2, VCAM1, hsCRP, and IL18, DuoSet ELISA (R&D Systems, Abingdon, United Kingdom) was used with a sensitivity of 93.8 pg/mL for E-Selectin, 625 pg/mL for MMP2, 15.6 pg/mL for VCAM1 and hsCRP, and 11.7 pg/mL for IL18. For measurement of IL6 and IL18BP, high-sensitivity Quantikine ELISA assays (R&D) were used with a sensitivity of 0.11 pg/mL for IL6 and 7.52 pg/mL for IL18BP.
Plasma E-selectin, matrix metalloproteinase (MMP)2, vascular cell adhesion molecule (VCAM)1, high-sensitivity C-reactive protein (hsCRP), interleukin (IL)18, IL18-binding protein (IL18BP) and IL6 levels were determined with ELISA. For E-Selectin, MMP2, VCAM1, hsCRP, and IL18, DuoSet ELISA (R&D Systems, Abingdon, United Kingdom) was used with a sensitivity of 93.8 pg/mL for E-Selectin, 625 pg/mL for MMP2, 15.6 pg/mL for VCAM1 and hsCRP, and 11.7 pg/mL for IL18. For measurement of IL6 and IL18BP, high-sensitivity Quantikine ELISA assays (R&D) were used with a sensitivity of 0.11 pg/mL for IL6 and 7.52 pg/mL for IL18BP.
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