Human umbilical vein endothelial cells (HUVECs) or HCAECs were seeded into sterile 6.5 mm Transwell inserts with a 0.4 μm pore polyester membrane (Corning; 3470) and cultured to confluency. A confluent endothelial monolayer was determined by transferring Transwell inserts to empty wells and refreshing cell culture medium in the top chamber with 500 μl endothelial cell growth medium; when media no longer permeated through to the lower chamber within 5 min, the endothelium was deemed intact. Post-treatment, 60 ng/mL streptavidin-HRP (R&D Systems; DY998) diluted in serum-free endothelial cell basal medium was added to the top chamber and the diffusion rate to the bottom chamber was assessed after 5 min. For detection of streptavidin-HRP, 20 μL cell culture medium was retrieved from the bottom chamber, to which 50 μL Substrate Reagent (R&D Systems; DY999) was added for 5 min. Subsequently, 25 μL Stop Solution 2N Sulfuric Acid (R&D Systems; DY994) was added. Spectrum absorption was measured at 450 nm using an ELISA plate reader.
Streptavidin hrp
Manufactured by R&D Systems
Sourced in United States, United Kingdom, France
Streptavidin-HRP is a conjugate of streptavidin, a protein derived from the bacterium Streptomyces avidinii, and horseradish peroxidase (HRP), an enzyme commonly used in immunoassays and other bioanalytical applications. The streptavidin-HRP conjugate binds to biotinylated molecules, allowing for the detection and quantification of target analytes in various experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Tmb substrate, by R&D Systems (6 mentions)
Tmb substrate, by BD (5 mentions)
Versamax microplate reader, by Molecular Devices (5 mentions)
Biotinylated anti mouse ige, by BD (4 mentions)
Spectramax m2 spectrometer, by Molecular Devices (4 mentions)
Clariostar plus, by BMG Labtech (4 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (4 mentions)
Collagenase d, by Merck Group (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Zymosan a, by Merck Group (3 mentions)
Tween 20, by Merck Group (3 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by R&D Systems (3 mentions)
Streptavidin horseradish peroxidase hrp, by R&D Systems (3 mentions)
Transwell plate, by Corning (3 mentions)
Biotinylated anti mouse ige or igg1, by BD (3 mentions)
Tmb substrate, by Thermo Fisher Scientific (3 mentions)
Protein g agarose, by Thermo Fisher Scientific (2 mentions)
Spectramax plus 384, by Molecular Devices (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (2 mentions)
151 protocols using streptavidin hrp
1
Transwell Assay for Endothelial Permeability
2
C5a/C5aR Binding and Phosphorylation Assay
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MSCs were seeded overnight at (3000 cells/cm 2 ) in 96-well microplates. Cells were incubated with conditioned media for 5 min. In parallel, cells were also pre-incubated for 5 min with or without 10 nM of W54011, a specific C5aR antagonist (Merck, Darmstadt, Germany). After washing, cells were fixed (3% paraformaldehyde, 20 min) and saturated (5% BSA, 1 h). C5a/C5aR binding was visualized by biotinylated mouse anti-human C5a Supernatants from injured PDL cells incubated with materials' extracts (conditioned media) were applied on MSCs cultures to evaluate C5a binding to MSCs C5aR and its phosphorylation by ELISA, to quantify their proliferation using MTT assay and their migration using Boyden chambers IgG (2 μg/mL, 2 h) followed by Streptavidin-HRP (20 min) and substrate solution (20 min) (R&D Systems, Lille, France). C5aR phosphorylation was visualized by biotinylated rabbit antihuman C5aR-p(Ser338) (Abcam, Paris France) (2 μg/mL, 2 h) followed by Streptavidin-HRP (20 min) and substrate solution (20 min) (R&D Systems, Lille, France). Optical densities were measured at 650 nm using an automatic microplate spectrophotometer (Metertech Inc., Taipei, Taiwan). C5a/C5aR binding and C5aR phosphorylation are expressed as percentage of the control condition.
3
Endothelial Cell Migration Assay
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Twenty-four well dishes with inserts (collagen-coated, 0.45-mm-meshed) were used for the assay. HDMECs (3 × 105 in 300 mL of endothelial growth medium) were seeded into each insert. Cells were cultured at 37 °C for 48 h to make a monolayer of the cells at the bottom. Then, CCR6 siRNA (final concentration, 10 nM) was added to the top chamber and cultured for another 48 h. Next, cells were stimulated with 100 ng/mL of recombinant human tumor necrosis factor-α (R&D systems, Minneapolis, MN, USA) for 24 h. After the stimulation, the bottom chambers were refilled with 1 mL of serum-free media, while serum-free media including 15 ml/mL of Streptavidin-HRP (R&D systems) were added in the top chambers. After 30-min incubation at 37 °C, 20 mL of media from the bottom chambers were transferred to a new 96-well plate in triplicate. Then, 50 mL of TMB substrate (Abcam) was added in each well and incubated for 5 min at room temperature. The reaction was stopped by adding 25 ml of stop solution (Abcam) into each well, and absorption at 450 nm was measured.
4
Measuring Serum HDM-specific IgE
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Serum HDM-specific IgE was measured by incubating plates overnight with 0.01% HDM in PBS and blocked with 1% BSA prior to addition of serum for 1 h. Serum was depleted of IgG by incubating with protein G agarose (ThermoFisher Scientific, Waltham, MA) overnight at 4° C prior to analysis. Following 6 washes with PBS plus 0.05% Tween-20, biotinylated anti-mouse IgE (Pharmingen, San Jose, CA) was added for 1 h. Plates were washed six times prior to addition of streptavidin-HRP (R & D Systems, Minneapolis, MN) for 30 min. TMB substrate was added for 30 min to determine the quantity of HDM-specific antibody.
5
Quantifying Plasma C5a Levels by ELISA
6
Enzyme-Linked Immunosorbent Assay for OxLDL
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Wells of a 96-well plate were coated overnight with 2 μg/100 μL of human OxLDL (AlfaAesar) in PBS. Wells were blocked with 200 μL of PBS-0.05% Tween + 1% BSA (Sigma-Aldrich, Oakville, ON, Canada) for 1 h. Wells were incubated at room temperature with diluted serum or BALF (1:3 × 106 and 1:10 [5 ], respectively, in PBS-0.05% Tween + 1% BSA) for 2 h. Wells were then washed 5 times with PBS-0.05% Tween. For detection, wells were incubated with the goat anti-mouse IgG + IgM + IgA H&L coupled to biotin (0.25 μg/mL in PBS-0.05% Tween + 1% BSA; AbCam, Toronto, ON, Canada) for 1 h, washed, incubated with Streptavidin-HRP (1:40; R&D systems, Minneapolis, MN, USA) for 30 min, washed and incubated with TMB substrate reagent BD OptEIA™ (BD Biosciences, San Jose, CA, USA) for 20 min. The reaction was stopped after 30 min with 2 N H2SO4 and the absorbance at 450 nm was read using Synergy H1 Hybrid Reader (BioTek, Winooski, VT, USA).
7
Assay for Oxidative Stress and Collagen Degradation
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Chemicals and reagents were obtained from the following commercial sources:
2',7'-dichlorodihydrofluorescein diacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), RPMI 1600 and an antibiotic solution containing 10,000 units of penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per mL were purchased from Thermo Fisher Scientific, Gibco (Grand Island, NY, USA). BlockAce was purchased from DS Pharma Biomedical (Japan), Biotin-HABP (biotinylated linker protein hyaluronan, 0.25mg/mL) was purchased from Hokudo Co. (Japan), Anti-Human Collagen type 1 antibody was purchased from Rockland Immunochemicals (US), Anti-human MMP-1, anti-IgG/HRP, and Streptavidin-HRP were purchased from R&D Systems (Japan), Can Get Signal was purchased from Toyobo Co. (Japan), EZ West blue dye was purchased from Atto Corp. (Japan), Chlorogenic acid (CGA) was purchased from Cayman Chemical (US). Fluorescein-5-thiosemicarbazide and sodium hyaluronate, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay reagent kit was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
2',7'-dichlorodihydrofluorescein diacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), RPMI 1600 and an antibiotic solution containing 10,000 units of penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per mL were purchased from Thermo Fisher Scientific, Gibco (Grand Island, NY, USA). BlockAce was purchased from DS Pharma Biomedical (Japan), Biotin-HABP (biotinylated linker protein hyaluronan, 0.25mg/mL) was purchased from Hokudo Co. (Japan), Anti-Human Collagen type 1 antibody was purchased from Rockland Immunochemicals (US), Anti-human MMP-1, anti-IgG/HRP, and Streptavidin-HRP were purchased from R&D Systems (Japan), Can Get Signal was purchased from Toyobo Co. (Japan), EZ West blue dye was purchased from Atto Corp. (Japan), Chlorogenic acid (CGA) was purchased from Cayman Chemical (US). Fluorescein-5-thiosemicarbazide and sodium hyaluronate, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay reagent kit was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
8
Quantitative GR Protein Assay
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MaxiSorp 96-well F-bottom plates (Thermo Fisher Scientific) were coated for 2 h with 5 µg/ml monoclonal GR antibody, followed by overnight blocking with 1% BSA in PBS-Tween (0.05% Tween 20, Sigma-Aldrich) at 4°C. After washing, 300 µg total protein lysate was added per sample. A 15x GR synthetic peptide (LifeTein) was used as a positive control. This peptide was serial diluted to create a standard curve (in duplo). All samples were measured both undiluted and diluted 2× and 4× with 0.1 M PBS. Samples and GR peptide were incubated on the plate for 1 h at room temperature. After washing, all wells were incubated for 1 h with biotinylated monoclonal anti-GR antibody at a final concentration of 0.25 µg/ml in PBS-Tween 20/1% BSA. After washing again, samples were incubated for 20 min with Streptavidin-HRP (R&D Systems) diluted 1:200 in PBS-Tween 20/1% BSA. Following extensive washing, samples were incubated with substrate reaction mix (R&D Systems) for 15 min and stopped using 2N H2SO4. Read-out was carried out using a plate reader (Varioskan) at 450 nm and 570 nm.
9
Quantitative ELISA for B7-H6 and BAG6 Proteins
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ELISA were performed as previously described [25 (link)]. Briefly, 96-well plates were coated with 5μg/mL mouse anti-B7-H6 mAb (clone 5.51.18) or anti-BAG6 mAb. B7-H6-Fc and BAG6 fusion protein were used as standard. The standard and the samples were added at 100μL/well, and the plates were incubated overnight at 4°C. After incubation, plates were washed and B7-H6 protein was detected with the biotinylated rabbit anti- B7-H6 796 antibody at a final concentration of 1 mg/mL in PBS/ 0.05% Tween20 for 2 hours at room temperature. After washing, streptavidin-HRP (1:200; R&D) was added for 30 minutes at room temperature. Subsequently, plates were washed and developed using 3,3′,5,5′-tetramethylbenzidine (TMB) Liquid Substrate System (Sigma). The absorbance was measured at 450 nm.
10
Cytokine and Mediator Secretion Analysis
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Supernatants from IEC/PBMC, IEC/moDC and ccDC/T-cell co-cultures were analyzed for cytokine and mediator secretion. Commercially available kits were used to determine IFNγ, IL-13, IL-17A (Thermo Fischer scientific, Waltham MA, USA), IL-10 (U-Cytech, Utrecht, The Netherlands), galectin-3, -4, -9 (R&D systems, Minneapolis, MN. USA) and IL-5 (Biolegend, San Diego, CA, USA) secretion according to the manufacturer’s protocol. Human galectin-4 or -9 were measured using antibody pairs (R&D systems). In short, high-binding Costar 9018 plates were incubated overnight at 4 °C with 0.75 µg/mL human galectin-4 or -9 affinity-purified polyclonal antibody. Non-specific binding was blocked with 1% BSA in PBS for 1 h, after which samples were incubated for 2 h at room temperature. After washing, biotinylated galectin-4 or -9 affinity-purified polyclonal antibodies (0.75 µg/mL) were added and incubated for 1 h. Then, plates were washed and streptavidin-HRP (R&D systems) was added and incubated for 1 h. After washing, tetramethylbenzidine was used as a substrate to develop the reaction (TMB, Thermo Fischer scientific), which was stopped with 1M H2SO4. Optical density was measured at 450 nm.
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