DNA isolation was performed using 7 ml aliquots of fresh or frozen whole blood (Fig 2 ; n = 192). Maxi DNA extraction kits from 5 different suppliers (GeneCatcher gDNA Kit from Invitrogen, QIAamp DNA Blood Maxi Kit from Qiagen, NucleoSpin Blood XL Kit from Macherey-Nagel, PerfectPure DNA Blood Kit from 5prime and Invisorb Blood Universal Kit from Stratec) as well as one published isopropanol precipitation protocol (IPP) [30 (link), 31 (link)] were used. DNA extraction kits used magnetic beads (Invitrogen), large (Marcherey-Nagel, Qiagen) and small (5prime) spin columns or precipitation (Stratec, IPP). DNA isolation was performed according to the manufacturers’ instructions and DNA was eluted in 300 μl (5prime kit), 600 μl (IPP), 1000 μl (Marchery-Nagel, Qiagen), 1400 μl (Stratec) or 1500 μl (Invitrogen). After elution, DNA was quantified using OD measurements performed in duplicates for each sample on a NanoDrop ND-1000 spectrophotometer (Fisher Scientific) and was stored at -20°C until further use. An overview about the characteristics of the kits can be found in Table 1 . Before qPCRs, samples were diluted to a DNA concentration of 10 ng/μl and quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) on a SpectraMax Paradigm Multi-Mode Microplate Detection Platform (Molecular Devices) before subsequent analyses. One sample was lost due to handling errors.
Spectramax paradigm multi mode microplate detection platform
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax Paradigm Multi-Mode Microplate Detection Platform is a versatile lab equipment designed to perform various microplate-based assays. It is capable of detecting absorbance, fluorescence, and luminescence signals from samples in microplates.
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Lab products found in correlation
Effectene transfection reagent, by Qiagen (2 mentions)
Prl tk, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Nucleospin blood xl kit, by Macherey-Nagel (1 mentions)
Invisorb blood universal kit, by Stratec (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Magnetic bead, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood maxi kit, by Qiagen (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Sensolyte 520 factor xa assay kit, by AnaSpec (1 mentions)
Atp bioluminescence assay kit, by Beyotime (1 mentions)
Amplex red glucose glucose oxidase kit, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
Salmon sperm dna, by Merck Group (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
17 protocols using spectramax paradigm multi mode microplate detection platform
1
Robust DNA Extraction from Whole Blood
2
Constructing Luciferase Reporter Vector
3
HCV NS3/4A Protease Inhibition Assay
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The HCV NS3/4A protease inhibition assay was carried out using the commercial SensoLyte® 520 HCV Protease Assay Kit (AnaSpec). The HCV NS3/4A protease is a 217 amino acid fusion protein (22.7 kDa) with NS4A co-factor fused to the N-terminus of NS3 protease domain. HCV NS3/4A protease activity was assessed by its ability to cleave the fluorogenic FRET peptide, and the subsequent unquenching by QXL 520 quencher of the 5-FAM fluorophore, which emits fluorescence. All extracts and controls were performed with three replicates in a 384-well plate format, each with a total reaction mixture of 18 μL. To begin, the test extracts and protease solution were incubated at room temperature for 10 min before the addition of the substrate. After substrate addition and gentle mixing the reaction was incubated at room temperature for one hour. The fluorophore was detected with the use of a SpectraMax® Paradigm® Multi-mode Microplate Detection Platform (Molecular Devices) by scanning at 490 nm excitation wavelength and 520 nm emission wavelength.
4
Factor Xa Inhibition Assay Protocol
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The Factor Xa inhibition assay was carried out using a commercial SensoLyte® 520 Factor Xa Assay Kit (AnaSpec). The Factor Xa activity was assessed by its ability to cleave the fluorogenic FRET peptide and the subsequent unquenching by QXL 520 quencher of the 5-FAM fluorophore, which emits fluorescence. All extracts and controls were performed with three replicates in a 384-well plate format, each with a total reaction mixture of 12.5 μL. To begin, the test extracts and Thrombin/Factor Xa solution were incubated for five minutes then the substrate was added. After substrate addition and gentle mixing the reaction was incubated at room temperature for one hour. The fluorophore was detected with the use of a SpectraMax® Paradigm® Multi-mode Microplate Detection Platform (Molecular Devices) by scanning at 490 nm excitation wavelength and 520 nm emission wavelength.
5
Mitochondrial Enzyme Activity Assay
6
Resazurin-based Cell Viability Assay
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We measured cell viability using resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide), a non-fluorescent blue dye that is reduced by viable cells to the highly fluorescent resorufin. The rate of reduction is directly proportional to cell fitness.66 (link) Resazurin was prepared as a stock solution of 75 μg/ml in calcium- and magnesium-free PBS and added to wells to give a final concentration of 10 μg/ml. Resorufin fluorescence was measured over 3 h using a microplate reader (SpectraMax Paradigm Multi-Mode Microplate Detection Platform, Molecular Devices LLC, Sunnyvale, CA, USA) by exciting at 530 nm and recording emission at 590 nm.
To obtain dose-response curves, we calculated resazurin reduction rates for each concentration of A2E or retinaldehyde by fitting the rate of resorufin appearance, from 40 min to 150 min, to regression lines (using GraphPad Prism, GraphPad Software, La Jolla, CA, USA). All regression fittings (over 1500 lines) gave R2≥0.99. For each plate, we normalized the reduction rates of the treated cells to control cells (empty liposomes or media alone).
To obtain dose-response curves, we calculated resazurin reduction rates for each concentration of A2E or retinaldehyde by fitting the rate of resorufin appearance, from 40 min to 150 min, to regression lines (using GraphPad Prism, GraphPad Software, La Jolla, CA, USA). All regression fittings (over 1500 lines) gave R2≥0.99. For each plate, we normalized the reduction rates of the treated cells to control cells (empty liposomes or media alone).
7
Fluorescence Polarization Assay for PLK Binding
8
Cytotoxicity of DHA and UDCMe-Z-DHA
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Cells were seeded into 96-well plates (3–5 × 103 cells/well), incubated overnight and then treated with 0–50 μM DHA or 0–10 μM UDCMe-Z-DHA for 24–72 h in HepG2 or Huh-7 cells, or treated with 0–100 μM DHA or UDCMe-Z-DHA for 72 h in NHDF cells. Cell viability was determined by the MTT assay and the absorbance was measured at 570 nm with 690 nm as a reference wavelength using the SpectraMax Paradigm Multi-Mode Microplate Detection Platform (Molecular Devices, San Jose, CA, USA). DMSO was used as the vehicle control for data normalization. IC50 values were calculated using GraphPad Prism software.
9
Measuring ATP Levels in U2OS and S180 Cells
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U2OS or S180 cells were cultured in 6-well plates (2 × 105 cells per well). After incubation over night, RPMI 1640 medium containing 3% FBS with or without serial concentrations of IS were replaced to the 6-well plates and incubated for 24 hrs after which, the level of ATP in the cells was measured using the ATP Bioluminescence Assay Kit (Beyotime, Haimen, China) following the manufacturer's instructions. Briefly, cells were harvested and lysed with a lysis buffer, followed by centrifugation at 10,000 × g for 5 min at 4°C. The level of ATP was determined by mixing 50 μL of the supernatant with 50 μL of luciferase reagent, which catalyzed the light production from ATP and luciferin. The emitted light was linearly related to the ATP concentration and measured using a Spectra-Max Paradigm Multi-Mode Microplate Detection Platform (Molecular Devices, Sunnyvale, California, United States) which was normalized to the protein concentration in each well. In SW1353 cells, the incubation medium was replaced by DMEM medium and ATP was assayed by the same method.
10
Quantifying PBD-Peptide Binding Kinetics
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