The amount of TGF-β3 release from the growth factor loaded cartilage ECMderived scaffold was determined via ELISA, as previously described [7, 22] . 96 well plates were coated with capture antibody (360 µg ml -1 ) with mouse anti-human TGF-β3 (R&D Systems, UK). The samples (8 time points) and TGF-β3 standards (ProSpec-Tany TechnoGene Ltd, Israel) were incubated for 2 hours. After washing and drying, detection antibody (18 µg ml -1 of biotinylated goat anti-human TGF-β3) was added to the plate and incubated (2 hours). The next step was to wash, dry and incubate the plate in streptavidin-HRP (horseradish-peroxidase) (R&D Systems, UK) for 20 minutes in the dark. Substrate solution (1:1 mixture of H 2 O 2 and tetramethylbenzidine; R&D Systems, UK) was added to each well, followed by incubation (20 minutes) avoiding direct light. Stop solution (2 N H 2 SO 4 ; Sigma-Aldrich, Germany) was added and the optical density was determined immediately with a plate reader set to 450 nm.
Streptavidin horseradish peroxidase hrp
Manufactured by R&D Systems
Sourced in United Kingdom
Streptavidin-horseradish peroxidase (HRP) is a conjugate composed of streptavidin, a protein that binds biotin, and horseradish peroxidase, an enzyme that catalyzes a colorimetric reaction. This conjugate is commonly used in various immunoassay and detection techniques that involve biotin-streptavidin binding.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin hrp, by R&D Systems (3 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by R&D Systems (3 mentions)
Versamax microplate reader, by Molecular Devices (3 mentions)
Tetramethylbenzidine, by R&D Systems (2 mentions)
Mouse anti human tgf β3, by R&D Systems (2 mentions)
Transwell insert, by Corning (2 mentions)
2 n h2so4, by Merck Group (1 mentions)
Substrate reagent, by R&D Systems (1 mentions)
Ezq protein quantitation kit, by Thermo Fisher Scientific (1 mentions)
Anti sdf 1 capture antibody, by R&D Systems (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Transwell plate, by Corning (1 mentions)
Rpeptide, by Stratech Scientific (1 mentions)
Mouse monoclonal anti α syn antibody, by BD (1 mentions)
Fluostar optima plate reader, by BMG Labtech (1 mentions)
8 protocols using streptavidin horseradish peroxidase hrp
1
Quantifying TGF-β3 Release from Cartilage Scaffold
2
TGF-β3 Quantification in ECM Scaffolds
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The TGF-β3 present in the ECM-derived scaffolds before seeding with FPSC was measured using a previously reported protocol 19 . Briefly, the scaffolds were treated with a solution of 4 M guanidine hydrochloride (Pierce) at 4°C for 2 days to extract any growth factors. TGF-β3 content was determined by ELISA 6c, [19] [20] . By following manufacturer instructions, capture antibody (360 µg ml -1 ) was in 96 well plates (mouse anti-human TGF-β3; R&D Systems, UK). The samples and standards (ProSpec-Tany TechnoGene Ltd, Israel) were incubated (2h). Furthermore, detection antibody (18 µg ml -1 of biotinylated goat antihuman TGF-β3) was added and incubated (2h). Next, the plate was washed, dried and incubated in streptavidin-HRP (horseradish-peroxidase; R&D Systems, UK). Substrate solution (1:1 mixture of H 2 O 2 and tetramethylbenzidine; R&D Systems, UK) was added to each well, followed by incubation (no light). Stop solution (2N H 2 SO 4 ; Sigma-Aldrich) was added and the optical density was determined (450 nm).
3
CXCL9 Peptides Compete with VEGF165 for HS Binding
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The ability of the CXCL9-derived peptides to compete with VEGF165 for HS binding was assessed in an ELISA-like GAG binding assay. First, the GAG binding plates (Galen Laboratory Supplies, North Haven, CT, USA) were coated with 25 µg/mL HS (Iduron, Alderley Edge, UK) in standard assay buffer (SAB) [100 mM NaCl, 50 mM sodium acetate, 0.2% (v/v) Tween® 20 in ultrapure water, pH 7.2] at room temperature overnight. The plates were washed with SAB and blocked with blocking buffer [SAB supplemented with 0.2% (w/v) gelatin] at 37 °C for 1 h to prevent nonspecific binding. Then, dilutions of VEGF165 and the peptides, CXCL9(74-103) or CXCL9(86-103), were added and allowed to interact at 37 °C for 2 h. Unbound material was removed through washing and biotinylated polyclonal goat anti-human VEGF165 antibody (PeproTech) in blocking buffer was added to interact with HS-bound VEGF165 at 37 °C for 1 h. Following additional washing steps, detection was performed with streptavidin-horseradish peroxidase (HRP) (R&D Systems) in blocking buffer at 37 °C for 30 min and conversion of 3,3’,5,5’-tetramethylbenzidine (TMB) to a colorimetric signal in the presence of 0.015% (v/v) H2O2. The reaction was stopped by the addition of H2SO4 and the absorbance was measured at 450 nm. All incubation steps were performed in the dark.
4
Quantification of Murine SDF-1α Levels
5
In Vitro Permeability Assay Protocol
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For the in vitro permeability assay, cells (2×105) were grown on a transwell insert (0.4 µm; Corning B.V. Life Sciences, The Netherlands) until a monolayer was formed. The upper chambers were reconstituted with 10% FBS-containing medium with MIF and inhibitors. At the indicated time points, the media in the upper chambers were replaced with 300 µl of serum-free media containing 4.5 µl of streptavidin-horseradish peroxidase (HRP) (R&D Systems, Inc., Minneapolis, MN). The media (20 µl) in the lower chambers were collected 5 min after adding streptavidin-HRP and assayed for HRP activity by adding 100 µl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (R&D Systems). Color development was detected using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.
6
Assessing HUVEC Barrier Permeability
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HUVECs (1× 105) were grown on the upper chambers of Transwell plates (0.4 μm; Corning, The Netherlands) until a monolayer was formed. The HUVECs monolayer was coincubated with BSA or NS1-treated washed platelets (1×107, 200 μl) along with Tyrode’s buffer. After the indicated time point, the upper chambers were reconstituted with 300 μl serum-free media, which contained 3 μl of streptavidin-horseradish peroxidase (HRP) (R&D Systems). Fifty microliters of medium in the lower chamber was collected 10 min after adding streptavidin-HRP and was assayed for HRP activity by adding 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (R&D Systems). The color development was detected by a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.
7
Transwell Permeability Assay for HUVEC Monolayer Integrity
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A Transwell permeability assay was performed as described in a previous study [58 ]. HUVECs (2 x 105) were grown on a Transwell insert (0.4 μm; Corning Life Sciences, Corning, NY, USA) until a monolayer formed. The upper chambers were reconstituted with 20 μg/ml NS1, culture supernatant from NS1-activated THP-1 cells, or the inhibitor-containing medium. After 24 h, the upper chambers were reconstituted with 300 μl of serum-free media containing 4.5 μl of streptavidin-horseradish peroxidase (HRP; R&D Systems, Inc., Minneapolis, MN, USA). Next, 20 μl of medium in the lower chamber was collected 5 min after the addition of streptavidin-HRP and was assayed for HRP activity by the addition of 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (R&D Systems). The color development at 450 nm was measured with a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).
8
Quantitative ELISA Assay for α-Synuclein
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Total α-syn level was determined by sandwich ELISA. Mouse
monoclonal anti-α-syn antibody (0.5 μg/ml; BD Biosciences, Oxford, UK)
was coated onto a NUNC Maxisorp 96-well plate overnight at RT. The plate
was washed in PBS/0.01% tween-20 and blocked for 1.5 hours in 1% BSA/PBS.
Tissue samples (insoluble and soluble extracts diluted 1:200 in PBS) were
added for two hours at RT with constant shaking. The plate was rinsed,
tapped dry and biotinylated polyclonal anti-α-synuclein (1 μg/ml; R&D
Systems, Oxford, UK) diluted in PBS was added for two hours at RT. The
plate was rinsed and tapped dry, streptavidin-horseradish peroxidase
(HRP) (1:200, R&D Systems) was added for 20 minutes and, after
further washing, chromogenic substrate (TMBS, R&D Systems) was added
for 20 minutes in the dark. The reaction was stopped with 2 N sulfuric
acid and absorbance at 450 nM read in a FLUOstar Optima plate reader (BMG
Labtech, Aylesbury, UK). Total α-syn levels were interpolated from
measurements made on serial dilutions of recombinant human α-syn ranging
from 62.5 to 0.98 ng/ml (rPeptide, Stratech, Suffolk, UK). Measurements
for each sample were repeated in duplicate.
monoclonal anti-α-syn antibody (0.5 μg/ml; BD Biosciences, Oxford, UK)
was coated onto a NUNC Maxisorp 96-well plate overnight at RT. The plate
was washed in PBS/0.01% tween-20 and blocked for 1.5 hours in 1% BSA/PBS.
Tissue samples (insoluble and soluble extracts diluted 1:200 in PBS) were
added for two hours at RT with constant shaking. The plate was rinsed,
tapped dry and biotinylated polyclonal anti-α-synuclein (1 μg/ml; R&D
Systems, Oxford, UK) diluted in PBS was added for two hours at RT. The
plate was rinsed and tapped dry, streptavidin-horseradish peroxidase
(HRP) (1:200, R&D Systems) was added for 20 minutes and, after
further washing, chromogenic substrate (TMBS, R&D Systems) was added
for 20 minutes in the dark. The reaction was stopped with 2 N sulfuric
acid and absorbance at 450 nM read in a FLUOstar Optima plate reader (BMG
Labtech, Aylesbury, UK). Total α-syn levels were interpolated from
measurements made on serial dilutions of recombinant human α-syn ranging
from 62.5 to 0.98 ng/ml (rPeptide, Stratech, Suffolk, UK). Measurements
for each sample were repeated in duplicate.
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