The concentrations of hIL-6, hBD1 and hBD2 were measured using ELISA development kits (Peprotech, Inc., London, UK) according to the manufacturer’s instructions. These kits contain the components required for the quantitative measurement of natural hIL-6, hBD2 and hBD2 in an ELISA sandwich format. Patient’s serum was added at a dilution of 1:2, 1:15 and 1:5 for hIL-6, hBD1 and hBD2 ELISA in order to fall within each kit’s measurement range (24–1500 pg/mL, 4–1000pg/mL and 16–2000 pg/mL, respectively). Standard curves were produced by serial dilutions of the kits’ hIL-6, hBD1 and hBD2 recombinant antigens.
Elisa development kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Israel
The ELISA Development Kit is a laboratory instrument designed to perform enzyme-linked immunosorbent assays (ELISA). It provides the necessary components and protocols to develop and optimize ELISA procedures for the detection and quantification of specific analytes in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Coomassie reagent, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg, by LI COR (2 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (2 mentions)
Donkey anti mouse igg, by LI COR (2 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (2 mentions)
β tubulin, by Cell Signaling Technology (2 mentions)
Synergy 2, by Agilent Technologies (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Dtx880, by Beckman Coulter (1 mentions)
Cytotox one homogenous membrane integrity assay, by Promega (1 mentions)
Dy999, by R&D Systems (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Ripa lysis buffer, by Keygen Biotech (1 mentions)
Ccl18, by Thermo Fisher Scientific (1 mentions)
2 2 azino bis 3 ethylbenzothiazoline 6 sulphonic acid, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Skanit software, by Thermo Fisher Scientific (1 mentions)
Varioskan, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ready set go elisa kit, by Thermo Fisher Scientific (1 mentions)
45 protocols using elisa development kit
1
Quantifying Cytokines and Defensins
2
Measuring Cytokine Levels in THP-1 Macrophages
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THP-1 cells were differentiated to THP-1 M0 macrophages in a 24-well plate (300,000 cells/well) and treated with 7-epi-clusianone for 72 h. The concentrations of tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) in the THP-1 M0 macrophages media were evaluated using human IL-6 and TNFα tetramethylbenzidine (TMB) enzyme-linked immunosorbent assay (ELISA) development kits (Peprotech, Rocky Hill, New Jersey) according to the manufacturer’s protocol. Colorimetric changes were measured using a SpectraMax 190 microplate spectrophotometer at 450 nm with wavelength correction set at 620 nm. Standard curves for each cytokine were run in parallel to convert the absorbance to concentration in each group.
3
Angiogenic Secretion Profiling of MSCs
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Mesenchymal stromal cells were cultured for 72 hours in the following 4 configurations with 2 variables: Coculture with U266, culture alone, under normoxia or hypoxia conditions. The secretions of angiogenic factors including VEGF, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) were measured after 72 hours. Medias conditioned by MSCs which is dealt with the 4 different methods mentioned above were collected and analyzed using commercially available human VEGF, PDGF, and bFGF enzyme-linked immunosorbent assay (ELISA) Development Kits (Peprotech, CA, USA).
4
Cytokine and Chemokine Analysis of MSC Cultures
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The concentrations of cytokines and chemokines were analyzed in the supernatants of MSC monoculture after 3 days incubation, by human IL-6 (Cat. No. 900-K16) and MCP-1 (Cat. No. 900-K31) ELISA development kits (Peprotech). Absorbance (405 nm, wavelength correction 650 nm) was measured using a Multimode Detector (DTX 880; Beckman Coulter) and results were quantified with standards.
5
Inflammatory Factors Detection via ELISA
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Medium supernatant and serum samples were collected for the measurement of inflammatory factors. Detection of IL-18 and IL-1βwas performed using the corresponding ELISA Development Kits (Peprotech, Rehovot, Israel) in accordance with the manufacturer’s instructions. The color development was monitored using an ELISA plate reader (iMark, Bio-Rad) at 405 nm with wavelength correction set at 650 nm.
6
Quantifying Human IL-6 and IL-8 Secretion
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Human IL-6 and IL-8 levels in cell culture supernatants were measured with ELISA Development Kits from PeproTech (Prague, Czech Republic) according to the manufacturer’s protocol.
7
Cytokine Quantification in Cell Culture
8
Lipigenine™ Stimulates HBD-1 Expression in Cell Culture
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EXAMPLE 4
Lipigenine™ was tested for its ability to stimulate an increase in HBD-1 concentration. The HBD-1 standard ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) ELISA development kits were obtained from PeproTech (Cat# 900-K202). ELISA were performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H2SO4 in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve.
The addition of Lipigenine™ showed high HBD-1 concentration at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-1 concentration of 20% was observed for 0.1% Lipigenine™ while an increase in HBD-1 concentration of 95% was observed for 1% Lipigenine™. These results are shown in
9
Lipigenine™ Stimulates HBD-1 Production
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Example 4
Lipigenine™ was tested for its ability to stimulate an increase in HBD-1 concentration. The HBD-1 standard AB TS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) ELISA development kits were obtained from PeproTech (Cat #900-K202). ELISA were performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H2SO4 in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve.
The addition of Lipigenine™ showed high HBD-1 concentration at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-1 concentration of 20% was observed for 0.1% Lipigenine™ while an increase in HBD-1 concentration of 95% was observed for 1% Lipigenine™. These results are shown in
10
Quantifying Inflammatory Cytokines in Microglia
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Blood samples were collected after MWM test and centrifuged at 2000×g for 20 min to obtain serum samples. Microglia culture medium was collected 24 h after LPS stimulation. Proteins from brains and primary cultured microglia were extracted using RIPA lysis buffer (Keygen Biotech. Co., LTD, Nanjing, China), and concentrations of protein were determined by the BCA Protein Assay Kit (Keygen Biotech. Co., LTD, Nanjing, China). All samples were stored at − 80 °C before assay. Detection of TNF-α, IL-1β, IL-6, and IFN-γ was performed by corresponding ELISA Development Kits (Peprotech, Rehovot, Israel) in accordance with the manufacturer’s instructions. Monitor color development with an ELISA plate reader (iMark, Bio-rad, Japan) at 405 nm with wavelength correction set at 650 nm.
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