HFF fibroblasts were cultured on the different surfaces studied, analyzing adhesion and proliferation using WST-1 (Roche Applied Science, Penzberg, Germany). This colorimetric determination quantifies cell activity by formazan staining. The mechanism is that mitochondrial dehydrogenases in living cells cause the separation of tetrazole salts, and the color of the soluble formazan is measured spectrophotometrically. The absorbance increases and can be correlated with increasing cell number. Cell viability was determined at the different specified culture times by incubating for 2 h with WST-1 1:10 in DMEM without serum and phenol red. The optical density (OD) at 440 nm of the cell supernatant was measured with the ELx800 universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Three different samples were studied for each surface type, and two different experiments were performed in parallel. The optical density (OD) at 440 nm of the cell supernatant was determined with the ELx800 universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Three samples were studied for each surface type, and two tests were performed. A curve was obtained using different numbers of cells from 3 × 103 to 50 × 103.
Elx800 universal microplate reader
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
The ELx800 Universal Microplate Reader is a compact and versatile spectrophotometric instrument designed for absorbance-based assays. It is capable of reading standard 96-well microplates.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 (cck8), by Dojindo Laboratories (5 mentions)
Cell counting kit 8 (cck8), by Beyotime (4 mentions)
P nitrophenyl phosphate substrate, by Southern Biotech (3 mentions)
Cck 8, by Dojindo Laboratories (3 mentions)
Kc junior software, by Agilent Technologies (2 mentions)
H5 mouse monoclonal antibody, by Southern Biotech (2 mentions)
Versamax microplate reader, by Molecular Devices (2 mentions)
Mouse albumin elisa quantitation set, by Fortis Life Sciences (2 mentions)
Mtt reagent, by Abcam (2 mentions)
Veritas microplate luminometer, by Promega (2 mentions)
M5655, by Merck Group (2 mentions)
Mtt cell proliferation assay kit, by Abcam (2 mentions)
Elisa plate, by Greiner (2 mentions)
Origin 9, by OriginLab (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Rapamycin, by Merck Group (1 mentions)
Methanol, by Fujifilm (1 mentions)
Acetone, by Fujifilm (1 mentions)
Ethanol, by Fujifilm (1 mentions)
Sulfuric acid, by Fujifilm (1 mentions)
126 protocols using elx800 universal microplate reader
1
Quantifying Fibroblast Adhesion and Proliferation
2
MTT Assay for Cell Viability
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MTT assay was used to determine the non-toxic concentrations of the inhibitors (generating a viability of over 80%). Exponentially growing cells were treated with trypsin and seeded into 96 well plates at 5 × 103 cells/well. After 72 h of cell culture, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The medium containing MTT was removed carefully and 150 μL of DMSO was added to each well. The plates were agitated until the dark blue crystal dissolved completely. The absorbance was measured using an ELx 800 Universal Microplate Reader (Bio-Tek, Inc. Winooski, Vermont) at wavelength of 490 nm. All MTT assays were performed with three repeated treatments and independently repeated three times. The survival rate was statistically analyzed by SPSS 20.0 (SPSS, Chicago, IL, USA), and the curves were generated by Origin 9.0. The data are expressed as the mean ± standard error of the mean (SEM). Student’s t-test was used for analyzing the differences between groups. Differences were statistically significant at P < 0.05. IC50 was analyzed according to the survival rate-concentration curve.
3
Rapamycin Inhibition of Trypomastigote Infection
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Cardiomyocytes were treated with different concentrations (from 0.625 to 5 μM) of rapamycin (Sigma) for 24 h. Then the cells were washed, plated and infected with β-galactosidase expressing trypomastigotes at a ratio of 10 parasites to 1 cell. After 2 h of infection, non-internalized parasites were washed out. After 48 h, monolayers were washed and assays were developed using CPRG as a β-galactosidase substrate as previously described (Faral-Tello et al., 2014 (link)), and quantified by measuring the absorbance at 570 nm using an ELx800 Universal Microplate Reader (BioTek Instruments Inc., Winooski, VT, United States). Wells with no drug were considered as the 100% of parasite replication.
4
Gas Chromatography-MS for DAP Metabolites
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Six DAP metabolites were measured by gas chromatography–mass spectrometry based on our previous method (Wu et al. 2006 ) with slight modification. Those measured were three dimethyl (DM) phosphate metabolites—dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP); and three diethyl (DE) phosphate metabolites—diethylphosphate (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The limits of detection (LODs) in our study were 0.5 μg/L for DMP, DMTP, DMDTP, and DEP, and 0.25 μg/L for DETP and DEDTP, defined as a signal-to-noise ratio of 3. The mean relative recoveries for six DAP metabolites ranged from 90.7% to 116.9%, with coefficients of variation (CV) ranging from 5.7% to 10.9%.
Creatinine concentrations were measured using ELx800 Universal Microplate Reader (wavelength 340–750 nm; BIO-TEK). Urinary DAP concentrations were presented both in μg/L and in μg/g Cre, adjusted based on creatinine levels.
Creatinine concentrations were measured using ELx800 Universal Microplate Reader (wavelength 340–750 nm; BIO-TEK). Urinary DAP concentrations were presented both in μg/L and in μg/g Cre, adjusted based on creatinine levels.
5
Analytical Techniques for Compound Characterization
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Ethanol (Wako), acetonitrile (Wako), mEthanol (Wako), sulfuric acid (Wako), acetone (Wako), trifluoroacetate (Tokyo Chemical Industry), dimethyl sulfoxide (Wako), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide), LPS and Griess reagent were all purchased from Sigma (St. Louis, MO, USA). An ELx 800 Universal Microplate Reader (BIO-TEK), VD-250R Freeze Dryer (TAITEC), US-105 Sonicator (SND), 5420 Centrifuge (IMOTO), R-300 Rotavapor (BUCHI), V-300 Vacuum Pump (BUCHI), High Performance Flash Chromatography (HPFC) system (Biotage AB), Medium Pressure Liquid Chromatography (MPLC) system (EPCLC, Yamazen), Preparative High Performance Liquid Chromatography (PHPLC) system (EPCLC, Yamazen), 1220 Infinity LC (Agilent Technologies), NMR spectrometer (Bruker DRX-600; Bruker Daltonics, Billerica MA, USA), Quadrupole time-of-flight (qTOF) mass spectrometer (Agilent Technologies, USA) and JASCO DIP-370 polarimeter (JASCO, Tokyo, Japan) were used.
6
Quantification of Gonococcal Antibodies
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Samples of vaginal wash and serum were collected from the mice at the indicated time points.17 , 26 (link) Gonococcus-specific IgA, IgG, IgM, and IgG subclass antibodies IgG1, IgG2a, IgG2b, and IgG3 in vaginal washes and sera were measured by ELISA on plates coated with whole gonococci, using undiluted vaginal wash and 10-fold diluted serum as starting dilutions. 17 , 26 (link) Total IgA, IgG, and IgM concentrations in secretions were assayed by ELISA on plates coated with anti-IgA, -IgG, or -IgM antibodies (Southern Biotech, Birmingham, AL). H5 mouse monoclonal antibody (specific for N. gonorrhoeae porin serovar PIB3) or affinity-purified mouse IgA, IgG, and IgM (Southern Biotech) were used to establish standard curves. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse IgA, IgG, IgM, IgG1, IgG2a, IgG2b, or IgG3 antibody (Southern Biotech) and p-nitrophenylphosphate substrate (Southern Biotech). Plates were read in a VersaMax microplate reader with SoftMax software (Molecular Devices, Sunnyvale, CA) or an ELX800 Universal microplate reader with KC Junior software (Bio-Tek Instruments, Winooski, VT). Antibody data were expressed as relative (fold increase) to the antibody levels detected in control samples (from sham-immunized mice) assayed simultaneously.
7
Quantification of Murine Immunological Markers
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Total urinary protein was determined using a Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories) according to the manufacturer’s instructions, and the urine was applied at dilutions of 1:100. Anti-IgG and anti-dsDNA IgG were analyzed using a mouse anti-IgG and anti-dsDNA IgG Kit (Bethyl Laboratories) according to the manufacturer’s instructions, and the sera were applied at dilutions of 1:100,000 and 1:500,000. Cytokines were analyzed using a mouse IL-18 and IL-1β ELISA Kit (R&D Systems) according to the manufacturer’s instructions. Absorbance at 450 nm was determined using an ELx-800 Universal Microplate Reader (BioTek).
8
Quantification of Immunomodulatory Factors
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IDO/IL-10/TGF-β in the plasma/PF were analyzed using ELISA kits (Wuhan EIAab Science; IDO analysis, and Affymetrix eBioscience; IL-10/TGF-β analysis). Proteins were quantified with ELX-800 Universal Microplate Reader (Bio-Tek, Winooski, VT) using Gen5™ software (Bio-Tek, Instruments, USA).
9
MTT Assay for Cell Viability
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Exponentially growing cells were seeded into 96 well plates at 5 × 103 cells/well. After 72 h of incubation, 20 μL of MTT (5 mg/mL) was added to each well. After incubation for an additional 4 h, the medium containing MTT was discarded and replaced with 150 μL of DMSO to dissolve the dark blue-purple crystal. The absorbance was measured at a wavelength of 490 nm, using an ELx 800 Universal Microplate Reader (Bio-Tek, Inc. Winooski, VT). The relative survival rate for the cells was analyzed using the SPSS 20 program (SPSS Inc., Chicago, IL) and the survival rate – drug concentration curves were generated using Origin 9.0 software (OriginLab Corporation, Northampton, MA). The concentration of drug required to inhibit cell viability by 50% (IC50 value) was determined using Origin 9.0 software.
10
Measuring Cell Metabolic Activity using MTT Assay
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To assess cell metabolic activity, a 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay (Sigma‐Aldrich) was utilised 20 . MTT was dissolved in PBS at 0.05 g/mL and aliquoted at 15 μL/well 8/24 hours post‐irradiation and incubated for 4 hours at 37°C. MTT solution was aspirated and replaced with 50 μL/well of dimethyl sulphoxide (Sigma‐Aldrich). Absorbance was read at 570 nm using a microplate reader (ELx800 Universal Microplate reader; Bio‐Tek Instruments, Winooski, Vermont).
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