The glucose/galactose assay was performed in HepG2 cells after treatment with TCM injections or ingredients as described previously (Swiss and Will 2011 (link); Kamalian et al., 2015 (link); Orlicka-Płocka et al., 2020 (link)). Briefly, HepG2 cells were seeded at a density of 1× 104 cells/well in 96-well plates and cultured in either galactose or glucose medium. After 48 h of treatment with various concentrations of compounds or DMSO vehicle, total cellular adenosine triphosphate (ATP) content was examined with the CellTiter-Glo™ Luminescent Cell Viability Assay Kit from Promega (Madison, WI, United States) according to the manufacturer’s protocol. And the luminescence signal was measured by the SpectraMax paradigm microplate reader from Molecular Devices (Sunnyvale, CA, United States). The IC50 values, defined as the drug concentrations producing a 50% reduction in cellular ATP content, were calculated by fitting the data to the “log (inhibitor) vs. response --Variable slope (four parameters)” equation.
Spectramax paradigm microplate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax Paradigm microplate reader is a multi-mode detection instrument designed for a variety of microplate-based assays. It provides absorbance, fluorescence, and luminescence detection capabilities in a compact, easy-to-use platform.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Dihydrorhodamine 123 dhr, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
M8180, by Solarbio (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Sytox green, by Corning (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Celltiter glo luminescent assay, by Promega (1 mentions)
Biomek nxp liquid handler, by Beckman Coulter (1 mentions)
Bright glo luciferase assay system, by Promega (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Fujifilm (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bright glo luciferase assay kit, by Promega (1 mentions)
Porcine pancreatic amylase, by Merck Group (1 mentions)
Starch solution, by Merck Group (1 mentions)
18 protocols using spectramax paradigm microplate reader
1
Glucose/Galactose Assay in HepG2 Cells
2
Measuring Oxidative Stress in BALF Cells
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As described above, BALF cells (2.5 × 105) were distributed into 96-well opaque plates (2.5 × 105/well) in Gln-free RPMI medium in the presence or absence of 500 µM Gln. The cells were then stimulated with LPS (20 μg/mL) or phorbol myristate acetate (PMA, 100 nM, Merck, Darmstadt, Germany) as a positive control. Then, dihydrorhodamine (DHR) 123 (1.2 μM; Sigma-Aldrich, St Louis, MO, USA) was added, and ROS production was measured every 10 min for 3 h at 35 °C, using 530/590 nm excitation/emission, on a Spectra-Max® Paradigm® microplate reader (Molecular Devices, San Jose, CA, USA).
3
Quantifying Inflammatory Markers in BALF
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The supernatants from each sample obtained after BALF centrifugation were distributed into 96-well opaque plates and cf-DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), read in a spectrofluorometer (Spectra-Max® Paradigm® microplate reader, Molecular Devices, Sunnyvale, CA, USA) at 485/538 nm excitation/emission [18 (link)].
To quantify elastase activity, supernatants (25 µL) were incubated with the elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (0.25 mM, Sigma) in buffer (50 mM HEPES, 100 mM NaCl, 0.01% Triton X-100) for 30 min at 37 °C and 5% CO2, protected from light. The reaction product was analyzed at 360/455 nm. MPO activity was assayed in BALF supernatants (7 µL) incubated with hexadecyltrimethylammonium bromide (HTAB, 60 µL), tetramethylbenzidine (TMB, 4 µL), and hydrogen peroxide (H2O2, 40 µL). After 60 min at 37 °C, the colorimetric reaction was interrupted by adding sodium acetate (2M). The reaction product was analyzed at 630 nm on a spectrophotometer [18 (link)].
To quantify elastase activity, supernatants (25 µL) were incubated with the elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (0.25 mM, Sigma) in buffer (50 mM HEPES, 100 mM NaCl, 0.01% Triton X-100) for 30 min at 37 °C and 5% CO2, protected from light. The reaction product was analyzed at 360/455 nm. MPO activity was assayed in BALF supernatants (7 µL) incubated with hexadecyltrimethylammonium bromide (HTAB, 60 µL), tetramethylbenzidine (TMB, 4 µL), and hydrogen peroxide (H2O2, 40 µL). After 60 min at 37 °C, the colorimetric reaction was interrupted by adding sodium acetate (2M). The reaction product was analyzed at 630 nm on a spectrophotometer [18 (link)].
4
Quantifying Cancer Cell Proliferation
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For MTT assay, 1 × 103 cancer cells were seeded in each well of 96-well plates, and each sample was performed in triplicate. After incubation, the plates were added with 20 μl per well of MTT solution (M8180, Solarbio) and incubated at 37 °C for 4 h. After aspiration of the supernatants, cells were incubated with 150 μl of dimethyl sulfoxide (Solarbio) at 37 °C for 30 min. The absorbance was measured at 490 nm in a SpectraMax Paradigm microplate reader (Molecular Devices). For EdU staining, cancer cells were pulse labeled with 50 μM of EdU in Dulbecco’s minimum essential medium for 2 h. Then cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and stained with the EdU Staining Proliferation Kit (UElandy). Cells were counterstained by Hoechst 33342 (UElandy) for nuclei staining. Images were captured under an Olympus microscope.
5
Quantifying Bacterial Membrane Permeability
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To quantify membrane permeabilization of E. coli and B. subtilis caused by the peptides the SYTOX green assay, adapted from Roth et al. was used [27] . Bacterial suspensions in the mid-logarithmic phase of growth were diluted to a cell density of 1x10 6 CFU/mL and incubated with SYTOX green (Life Technologies, South Africa) at a final concentration of 0.1 µM in 10 mM sodium phosphate (NaP) buffer pH 7.4 for 15 minutes at 37˚C in a shaking incubator. The bacteria and SYTOX green mixture was then exposed to a concentration range of Mel, Os and Os-C (0.1 -10 µM) for 1 hour at 37˚C in a shaking incubator in a black, flat-bottom, polystyrene costar 96-well plate (Corning, New York, USA). The plate was then transferred to the SpectraMax Paradigm microplate reader (Molecular Devices, California, USA) and the fluorescence of each well measured using an excitation of 488 nm and emission of 530 nm. The data is presented as mean ± standard error of mean. Multiple comparisons were tested by one-way ANOVA followed by the Tukey's post hoc test (http://statistica.mooo.com/).
6
Riboglycated BSA Cytotoxicity Assay
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Cell viability was detected using a cell counting kit-8 assay (CCK-8) (Dojindo, Japan). The SH-SY5Y cells were plated in 96-well plates at a concentration of 104 cells per well. After 24 h, different concentrations of riboglycated BSA (0, 0.6, 1.2, 1.5, 1.8, 2.4, and 3 μM) were added to SH-SY5Y cells followed by measurements of cell viability after 24 h. In another experiment, 1.5 μM riboglycated BSA was added to cells 24 h after plating for different time intervals (0, 6, 12, 24, 36, 48, 60, and 72 h) followed by measurements of cell viability. After treatment, the CCK-8 reagent was added, and the plates were incubated at 37°C for 1.5 h. Under the same conditions, the cells were treated with glucoglycated BSA in addition to the ribose, glucose, BSA, and Tris–HCl controls. The absorbance was recorded at 450 nm using a SpectraMax Paradigm microplate reader (Molecular Devices, United States).
7
Amyloid Fibril Formation Assay
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Purified protein (50 μM) in a buffer of 25 mM Tris–HCl (pH 8.0 for RRM1 or pH 9.0 for RRM1‐RRM2) and 100 mM NaCl was centrifuged at 20,000g for 10 min and filtered through a 0.22‐μm Millex‐GV filter to remove any insoluble material or aggregates. The samples were then shaken at 200 rpm at 37°C for 3 days to promote fibril formation. The freshly‐formed fibrillar solutions (5 μL) were subjected to ThT‐binding assays. ThT amyloid binding dye (ThT, purchased from Sigma) was dissolved in water to make a stock solution of 1 mM, which was filtered through a 0.2‐μm Sartorius Minisart syringe filter and stored in 1 ml aliquots at −20°C and protected from light. The fibrillar solutions were mixed with ThT dye to a final concentration of 20 μM dye and ~10 μM protein. After 5‐min incubation in the dark, the samples were excited at 438 nm and the fluorescence emission intensity was recorded from 472 to 600 nm on a SpectraMax Paradigm microplate reader (Molecular Devices).
8
Genome Editing of Liver Organoids
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Human liver organoids were transfected with 1800 ng of px459 plasmid (Cas9 plus sgRNA) using Lipofectamine 3000 (Thermo Fisher Scientific) as previously reported (42 (link)). In brief, single-cell suspensions at 80–90% confluency of organoids and the DNA:Lipofectamine 3000 mixture were plated and centrifuged at 32°C at 500 x g for one hour. Five hours after incubation, cells were seeded in 24-well plates at 80% confluency on 50 μl BME matrix. Organoids were treated with 40 μM nutlin for 6 days; then their viability was measured using a CellTiter-Glo luminescent assay based on quantitation of ATP according to the manufacturer's instructions (Promega). Luminescence was measured using a SpectraMax®Paradigm® microplate reader (Molecular Devices).
9
High-Throughput Neutralizing Antibody Assay
10
Quantitative Amyloid Fibril Assay
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Lyophilized
insulin was dissolved in HCOOH (pH 3.0) at a concentration of 6 mg
mL–1 and filtered using 50 kDa Amicon Ultra centrifugal
filters (Millipore, prod. no. UFC505096). The protein was dissolved
to a final concentration of 2 μM in 0.75 M D-mannitol, 1 M GdmCl,
10 μM ThT, HCOOH (pH 3.0) in a reaction volume of 28.5 μL
per well. To each reaction, either 1.5 μL of serial 10-fold
dilutions from 104 to 1015 of the standard fibril
sample (dissolved in HCOOH, pH 3.0) or HCOOH (pH 3.0, negative control)
was added. Each dilution and the control were performed in 16 technical
replica on black 384-well polystyrene microplates (Corning, prod.
no. 3540) covered with sealing tape (Sarstedt, prod. no. 95.1999).
Microplates were incubated at a constant temperature of 30 °C
with cyclic agitation (1 min at 300 rpm followed by 2 min quiescent)
on a SpectraMax Paradigm microplate reader (Molecular Devices). To
monitor the aggregation kinetics, ThT fluorescence was measured at
482 nm following excitation at 440 nm every 3 min from the bottom
of the microplate. The error of the assay was calculated from three
independent replicate measurements of the microplate assay (for data
analysis see sectionDigital Data Analysis ). The replicate measurements are shown in Figure 5 and Figure S3 .
insulin was dissolved in HCOOH (pH 3.0) at a concentration of 6 mg
mL–1 and filtered using 50 kDa Amicon Ultra centrifugal
filters (Millipore, prod. no. UFC505096). The protein was dissolved
to a final concentration of 2 μM in 0.75 M D-mannitol, 1 M GdmCl,
10 μM ThT, HCOOH (pH 3.0) in a reaction volume of 28.5 μL
per well. To each reaction, either 1.5 μL of serial 10-fold
dilutions from 104 to 1015 of the standard fibril
sample (dissolved in HCOOH, pH 3.0) or HCOOH (pH 3.0, negative control)
was added. Each dilution and the control were performed in 16 technical
replica on black 384-well polystyrene microplates (Corning, prod.
no. 3540) covered with sealing tape (Sarstedt, prod. no. 95.1999).
Microplates were incubated at a constant temperature of 30 °C
with cyclic agitation (1 min at 300 rpm followed by 2 min quiescent)
on a SpectraMax Paradigm microplate reader (Molecular Devices). To
monitor the aggregation kinetics, ThT fluorescence was measured at
482 nm following excitation at 440 nm every 3 min from the bottom
of the microplate. The error of the assay was calculated from three
independent replicate measurements of the microplate assay (for data
analysis see section
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