Cell culture media supernatants from undifferentiated or differentiated cells across several timepoints (see Endoderm differentiation) of different cell lines were collected, spun at 300 × g, 5 min at 4 °C. Cell free supernatants were again collected and snap frozen at −80 °C in dry ice. Prior to ELISA, supernatants were thawed on ice and prediluted 1:200 in reagent diluent (R&D Systems, DY995) of the Human Dkk4 DuoSet ELISA KIT (R&D Systems, DY1269). DKK4 ELISA has been carried out according to the manufacturer’s instructions. Cell culture media supernatants from undifferentiated or differentiated cells across several timepoints (see. Endoderm differentiation) of different cell lines were collected, spun at 300 × g, 5 min at 4 °C. Cell free supernatants were again collected and snap frozen at −80 °C in dry ice. Prior to ELISA, supernatants were thawed on ice and prediluted 1:200 in reagent diluent (R&D Systems, DY995) of the Human Dkk4 DuoSet ELISA KIT (R&D Systems, DY1269). DKK4 ELISA has been carried out according to the manufacturer’s instructions. HRP raw values were measured on the GloMax-Multi Detection System (Promega).
Dy995
Manufactured by R&D Systems
The DY995 is a laboratory instrument designed for measuring the electrical impedance of biological samples. It operates by applying a small alternating current to the sample and measuring the resulting voltage, allowing for the calculation of the sample's impedance. This data can be used to analyze the properties of the sample, such as its composition and structure. The DY995 is a versatile tool that can be used in a variety of research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Wa126, by R&D Systems (7 mentions)
Dy999, by R&D Systems (6 mentions)
Dy994, by R&D Systems (6 mentions)
Dy992, by R&D Systems (6 mentions)
Dy006, by R&D Systems (6 mentions)
Dy990, by R&D Systems (6 mentions)
Dy268, by R&D Systems (6 mentions)
248 n4 250 cf, by R&D Systems (4 mentions)
Bicinchoninic acid (bca), by Abcam (2 mentions)
Filtered centrifree ultra ltration device, by Merck Group (2 mentions)
Glomax multi detection system, by Promega (1 mentions)
Human dkk4 duoset elisa kit, by R&D Systems (1 mentions)
Dy2150, by R&D Systems (1 mentions)
Dy460, by R&D Systems (1 mentions)
Dy1588 05, by R&D Systems (1 mentions)
248 bdb 250 cf, by R&D Systems (1 mentions)
Microplate spectrophotometer, by Agilent Technologies (1 mentions)
11 protocols using dy995
1
Quantification of Dkk4 in Cell Differentiation
2
Measuring Murine Complement C3a and C5a
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Murine complement C5a levels were determined using the mouse complement component C5a duoset and accompanying standard from R&D systems (catalog #DY2150; Minneapolis, MN). An ELISA for murine complement C3a was established using a murine C3a standard and antibodies from BD Biosciences (Mississauga, Canada). The capture rat monoclonal anti-mouse C3a antibody (catalog # 55820, clone: I87-1162) was coated overnight onto 96-well plates in 100 μL of PBS at a concentration of 2 μg/mL. Wells were washed × 3 with wash buffer (R&D catalog #WA126) followed by blocking for 2 h with 300 μl of reagent diluent (R&D systems catalog #DY995). Plasma samples in duplicate were diluted 1/50 and 1/125 in sample diluent to a final volume of 100 μl and incubated for 2 h. After 3 washes, 100 μl of the biotinylated detection monoclonal rat anti-mouse C3a antibody (clone I87-419, catalog #55821) 0.5 μg/mL in reagent diluent was incubated for 1 h. Wells were washed × 4 and incubated for 20 min at room temperature with 100 μl of streptavidin-biotinylated horseradish peroxidase (1:3000), followed by 2 washes, and development with the substrate solution containing o-phenylenediamine using a plate reader set to 450 nm. A standard curve was generated with purified murine C3a (catalog # 558618). The sensitivity range of the assay was 0.1 nM to 2.5 nM. Intra-assay and inter-assay precision was 8–10%.
3
Quantification of Recombinant BDNF
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Filtered (centrifree ultrafiltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-N4-250/CF, R&D Systems, Canada) of known concentrations (typically 250 mgL−1) in the phosphate buffered saline (PBS) pH 7.4 ± 0.2, 0.15 M (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration in the solvent. To minimize errors in concentration measurements, two complementary spectrophotometric techniques were used: the BCA (protein quantification bicinchoninic acid assay, kit for low concentration, ABCAm, Cambridge, UK) and UV absorbance at 280 nm measured with microplate spectrophotometer (BioTek Epoch, United States). Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01–2 mgL−1. The exact concentration of these solutions after membrane filtration was determined by commercially available Enzyme-Linked Immunosorbent Assay (ELISA) (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298 ± 0.1 K.
4
Protein Loading Profiling of PAMAM Nanoparticles
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Protein loading profile at PAMAM-based nanoparticles and PEGylated PAMAM-based nanoparticles with negative charge core as well as positive charge core were conducted by solution depletion ELISA technique (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). Afterward, to determine unbound BDNF molecules more accurately after adsorption at PAMAM particles, a two-step Laser Doppler Velocimetry (LDV) technique with the aforementioned Malvern device's aid was used. LDV method exploiting the calibrating measurements uses various colloid particles (in our study modeled latex microparticles) for efficient monitoring of desorbed protein molecule concentration to determine maximum protein coverage at PAMAM nanoparticles.
5
Dual-Crosslinked Hydrogels for Delivery
6
ELISA for Quantifying IgG Isotypes
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ELISA assays were carried out for IgG1 and IgG3. Two plates (provided with the kits) were coated with capture antibody diluted in sterile PBS and incubated overnight at 4 °C. Following 3 washes with wash buffer 0.05% Tween 20in PBS, plates were blocked with 250 µL blocking buffer for 1 h with 200 μL/well reagent diluent (R&D Systems, DY995). The serial dilutions of serum test samples, from untreated animals were prepared in reagent diluent and incubated with 50 μL/well overnight at RT. The plates were washed 4 times followed by addition of 100 μL HRP-labeled mouse IgG1and IgG3 diluted (1:100) in the reagent diluent to each and every well and incubated for 2 h. Plates were then washed 4 times followed by the incubation for 20 min with streptavidin-horseradish peroxidase (1:400) diluted in the reagent diluent, and 100 μL was added to all wells. Plates were washed 6 times and substrate was added and allowed to stand for 15 min for the development of color. The enzymatic color development reaction was terminated using stop solution (2N H2SO4), and plate was read at 450 nm with optical correction at 570 nm. The representative test curves for IgG2a and IgG1 with the absorbance values were compared to the total serum Ig levels obtained and were plotted as arbitrary units (AU).
7
Quantification of Circulating IGFBP-1 in Mice
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Circulating mouse IGFBP-1 was detected with an in-house–developed, sandwich ELISA using a commercially available kit (DY1588-05; R&D Systems). The capture antibody was coated on a flat-bottom Microlon 600 high-binding 96-well plate, (82050–720; VWR International) at 4 µg/ml in 100 µl/well of PBS overnight at 4°C. Samples of plasma were added at a concentration of 1:40, 5 µm in 195 µl of reagent diluent (DY995; R&D Systems). Samples and the standard were added to the coated plate and left to incubate overnight at 4°C. For the detection phase, we used the Bt polyclonal antibody raised against the mouse IGFBP-1 supplied by the kit (DY1588-05; R&D Systems) at a concentration of 400 ng/ml. The colorimetric detection reaction, termination, and plate reading were performed using the method described for the N3ECD.
8
Recombinant Neurotrophic Factor Preparations
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In our studies, unfiltered stock solutions of carrier-free recombinant human BDNF (rhBDNF) (248-BDB-250/CF, R&D Systems, Minneapolis, MN, USA) as well as carrier-free recombinant human NT3 (rhNT3) (248-N4-250/CF, R&D Systems), of known concentrations (typically 250 mg L−1), were prepared in phosphate buffered saline (PBS) pH 7.4 ± 0.2, 0.15 M (Biomed, Lublin, Poland) and stored for no longer than 2 months at a temperature of −20 °C. rhBDNF and rhNT3 are hereinafter collectively referred to as neurotrophins (NTs). Before each measurement, the stock solution was diluted to the desired bulk concentration, typically 25 mg L−1. The exact concentration of these solutions was determined by the commercially available enzyme-linked immunosorbent assay (ELISA) assay (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of the experiments was kept at a constant value equal to 298 ± 0.1 K.
9
Quantification of Recombinant Human BDNF
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Filtered (centrifree ultra ltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-N4-250/CF, R&D Systems, Canada) of known concentrations (typically 250 mg L -1 ) in the phosphate buffered saline (PBS) pH 7.4 +/-0.2 (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration in the solvent. To minimize errors in concentration measurements, two complementary spectrophotometric techniques were used: the BCA (protein quanti cation bicinchoninic acid assay, kit for low concentration, Abcam, kraj) and UV absorbance at 280 nm measured with microplate spectrophotometer (BioTek Epoch, United States).
Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01-2 mg L -1 . The exact concentration of these solutions after membrane ltration was determined by commercially available ELISA assay (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298 ± 0.1K.
Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01-2 mg L -1 . The exact concentration of these solutions after membrane ltration was determined by commercially available ELISA assay (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298 ± 0.1K.
10
BDNF Protein Standardization Protocol
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Filtered (centrifree ultra ltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-N4-250/CF, R&D Systems, Canada) of known concentrations (typically 250 mgL -1 ) in the phosphate buffered saline (PBS) pH 7.4 +/-0.2, 0.15M (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration in the solvent. To minimize errors in concentration measurements, two complementary spectrophotometric techniques were used: the BCA (protein quanti cation bicinchoninic acid assay, kit for low concentration, ABCAm, Cambridge, UK) and UV absorbance at 280 nm measured with microplate spectrophotometer (BioTek Epoch, United States). Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01-2 mgL -1 . The exact concentration of these solutions after membrane ltration was determined by commercially available Enzyme-Linked Immunosorbent Assay (ELISA) (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298±0.1K.
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