Inhibitory effects of the investigated compounds were determined using Universal IDO1/IDO2/TDO Inhibitor Screening Assay Kit from BPS Bioscience, Inc. (San Diego, CA, USA). This colorimetric assay is based on the measurement of the ability of IDO1, IDO2 and TDO to convert L-tryptophan into N-formylkynurenine (NFK). The experiments were performed according to the manufacturer’s guidelines. The final concentration of the compounds in the reaction mixture was 50 µg/mL. The amount of NFK was measured spectrophotometrically at 320 nm using Epoch BioTek microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The samples were run in triplicate and the results were expressed as mean ± SD.
Epoch biotek microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Epoch BioTek microplate reader is a compact, high-performance instrument designed for absorbance-based microplate assays. It measures the optical density of samples in microplates, enabling researchers to quantify a wide range of biological and chemical analytes.
Automatically generated - may contain errors
Lab products found in correlation
Universal ido1 ido2 tdo inhibitor screening assay kit, by BPS Biosciences (2 mentions)
Brain derived neurotrophic factor (bdnf), by R&D Systems (1 mentions)
Catalase, by Cayman Chemical (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Tissue master 125 homogenizer, by Omni International (1 mentions)
Glutathione peroxidase gpx, by Merck Group (1 mentions)
Tbars, by Cayman Chemical (1 mentions)
Superoxide dismutase sod, by Merck Group (1 mentions)
Endothelin 1 quantikine elisa kit, by R&D Systems (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Cck 8 solution, by Beyotime (1 mentions)
10 protocols using epoch biotek microplate reader
1
Colorimetric Assay of IDO Inhibition
2
Oxidative Stress and BDNF Measurement
3
Quantification of Plasma Endothelin-1 Using ELISA
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Plasma ET-1 levels were measured using a colorimetric enzyme-linked immunosorbent assay (Endothelin-1 Quantikine ELISA Kit, R&D Systems, USA & Canada). Briefly, 75 μL of standard or sample was added to each microplate well coated with ET-1 primary antibody and incubated for 1 hour on a shaker at room temperature. Following washing, 200 μL of ET-1 conjugate was added to each well and incubated for 3 hours at room temperature on the shaker. After washing, wells were incubated with 200 μL of substrate solution followed by addition of 50 μL of stop solution. Optical density of each well was determined at 450 nm using an Epoch Biotek microplate reader (BioTek, Winooski, VT, USA).
4
Serum Lipid Profile Determination
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Blood samples were taken after 8–12 h fasting, half sample was used for DNA isolation while the rest half was used to obtain serum. Serum was separated by centrifuging gel vacutainers at 10000 r.p.m. for 10 min, collected in sterilized Eppendorf, and screened for any infectious agents (HBV, HCV, HIV). Any positive samples were discarded and safe samples were used for the lipid profile determination. Serum TC, TG, HDL-c, and LDL-c were measured using commercially available kits (Spectrum Diagnostics, Egypt). Epoch, Biotek microplate reader (BioTek Instruments, Highland Park) was used for all optical density measurements.
5
Screening Assay for IDO Enzyme Inhibition
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Inhibitory effects of the investigated extracts and standards (salazinic acid, evernic acid, (−)-usnic acid), as well as the reference IDO1 inhibitor (epacadostat) were determined using Universal IDO1/IDO2/TDO Inhibitor Screening Assay Kit from BPS Bioscience, Inc. (San Diego, CA, USA). This colorimetric assay is based on the measurement of the ability of IDO1, IDO2 and TDO to convert l -tryptophan into N-formylkynurenine (NFK). The experiments were performed according to the manufacturer’s guidelines. The final concentration of the compounds and extracts in the reaction mixture was 100 µg/mL. The amount of NFK was measured spectrophotometrically at 320 nm using an Epoch BioTek microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The samples were run in triplicate and the results were expressed as mean ± SEM.
6
Genotyping of Cardiovascular Genes
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The pediatric blood samples were taken in EDTA vials and preserved at -20 °C. Genomic DNA from the human leukocytes/epithelial cells was isolated using the salting out method (see supplementary data for salting out method). The quality of extracted DNA was evaluated using an Epoch Biotek micro-plate reader (Biotek Instruments, USA) and DNA concentration was adjusted to 10 ng/uL. The SNPs of ISL1: rs1017 (NG_023040.1:g.16138A > T), NFATc1: rs7240256 (NG_029226.1:g.23449 T > C), VEGF: rs36208048 (NG_008732.1:g.3877C > A), TBX5: rs11067075 (NG_007373.1:g.51682G > T), and MTHFR: rs1801133 (NG_013351.1:g.14783C > T) genes were genotyped by using tetra primer the ARMS PCR and PCR–RFLP techniques. For primers sequence and restriction enzyme, see supplementary data (supplementary table 1 ).
7
Blood Lipid Profiling Protocol
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Blood samples were taken after 8-12 hr fasting, and half sample was used for DNA isolation, while the rest half was used to obtain serum. Serum was separated by centrifuging gel vacutainers at 10,000 rpm for 10 min, collected in sterilized Eppendorf, and screened for any infectious agents (HBV, HCV, and HIV). Any positive samples were discarded, and safe samples were used for lipid profile determination. Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) were measured using commercially available kits (Spectrum Diagnostics, Egypt). Epoch BioTek microplate reader (BioTek Instruments, Highland Park) was used for all optical density measurements.
8
Cell Viability Assay for H1299 and HCC827 Cells
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H1299 cells (4 ×103/well) and HCC827 cell (3 ×103/well) were seeded in 96-well plates. Five repetitive wells were set for each experiment. Cell activity was tested by cell counting kit-8 (CCK-8) (Dojindo, Kyushu, Japan) for 4 days. 10 µl CCK-8 was added into each well and then incubated with the cells for 2 h at 37 °C. The absorbance was recorded at 450nm with Bio Tek Epoch Microplate Reader (Bio Tek Instruments, Inc). The experiments were repeated 3 times.
9
Intracellular Superoxide Dismutase Quantification
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Intracellular superoxide dismutase levels were measured by using superoxide dismutase kits (Beyotime, China), following the manufacturer’s instructions. The data were measured by using a BioTek® Epoch microplate reader (BioTek Instruments, Inc. United States).
10
Cell Viability Assay via CCK-8
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Cell viability was measured by using the Cell Counting Kit-8 (CCK-8). In brief, after 24 h of treatment, cells were incubated with CCK-8 solution (Beyotime, China). To facilitate the reaction, stained cells were incubated at 37°C for 3 h. At the end of the treatment, the absorbance at 450 nm was measured using a BioTek® Epoch microplate reader (BioTek Instruments, Inc. United States).
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