Cells seeded in 96-well black plates were transfected with ZIKV NS4A-encoding plasmid at varying concentrations. After 24 h, cells were incubated with fluorescent dye tetramethylrhodamine methyl ester (TMRM) (Thermo Fisher Scientific) (20 nM, 30 min) at 37°C and washed with Dulbecco’s phosphate-buffered saline (DPBS). TMRM fluorescence was measured using Varioskan microplate reader (Thermo Fisher Scientific).
Varioskan microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Finland, Canada
The Varioskan microplate reader is a versatile instrument designed for a wide range of photometric measurements. It is capable of performing absorbance, fluorescence, and luminescence detection in microplates. The device can be used for various applications, including cell-based assays, enzyme activity, and DNA/protein quantification.
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Lab products found in correlation
Collagen based invasion assay kit, by Merck Group (3 mentions)
Mitosox, by Thermo Fisher Scientific (2 mentions)
Phenol red, by Thermo Fisher Scientific (2 mentions)
Pdt edl 1, by Hamamatsu Photonics (2 mentions)
Primescript rt master mix kit, by Takara Bio (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Gsh glo assay, by Promega (1 mentions)
Lactate glo assay, by Promega (1 mentions)
Glutamate glo assay, by Promega (1 mentions)
Glucose glo assay, by Promega (1 mentions)
Mouse crp elisa kit, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (1 mentions)
Caspase glo 3 7 assay system, by Promega (1 mentions)
Cytation 3 image reader, by Agilent Technologies (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Calcein am, by Abcam (1 mentions)
Hoechst 33258, by Abcam (1 mentions)
77 protocols using varioskan microplate reader
1
Analyzing Mitochondrial Membrane Potential
2
Mitochondrial ROS Detection using MitoSOX
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Mitochondrial ROS was detected using the fluorescence indicator MitoSOX (Life Technologies Inc.). RGK36 and RGK45 cells were incubated at 37 or 42 °C for 1 h and then incubated for 30 min in 5 μM MitoSOX diluted with HBSS. After incubation, the cells were washed three times with HBSS. Fluorescence intensity of MitoSOX was measured by a Varioskan micro plate reader (Thermo Fisher Scientific K.K., Kanagawa, Japan). The measurement wavelengths of excitation and emission were 510 and 580 nm, respectively.
3
Ovarian Cancer Cell Proliferation Assay
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Ovarian cancer cell Proliferation was determined by adding 10 μl of the WST-1 reagent to control and transfected ovarian cancer cells which were plated in the different wells of a 96 well plate (dilution of WST-1 to media 1:10). The cells were then incubated with WST-1 for 2 h in the incubator (37 °C and 5% CO2). Cell proliferation was finally quantified at 450 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA).
4
GDPase Activity Assay for ARF1
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[Δ17]ARF1 and ARNO-Sec-7 were subcloned into pET15 vectors (Novagen) as described previously[13 (link)21 (link)22 (link)23 (link)36 (link)]. N-Terminal truncated [Δ17]ARF1 (amino acids 18-181), lacking the first 17 amino acids and cytohesin-1-Sec-7 were expressed in Escherichia coli and purified by Ni-NTA chromatography (Ni-NTA agarose, Quiagen). GDP/GTP exchange was measured on [Δ17]ARF1 by tryptophan fluorescence because a large increase in intrinsic fluorescence of ARF occurs upon exchange of GDP for GTP[34 37 (link)]. All measurements were performed in PBS pH 7.4, 3 mM MgCl2 at 37°. [Δ17]ARF1 (1 μM) in PBS without MgCl2 was preincubated with GDP (80 μM) in the presence of EDTA (2 mM) for 15 min. The bound GDP was stabilized by addition of MgCl2 (final concentration 3 mM) and incubation for 5 min. For each exchange reaction 250 nM [Δ17]ARF1 was mixed with 10 nM ARNO-Sec-7 (total volume 200 μl) in the absence or presence of inhibitor. The reaction was started by injection of GTP (50 μM). The tryptophan fluorescence was measured at an excitation and emission wavelength of 280 and 340 nm, respectively. All fluorescent measurements were performed with a Varioskan microplate reader (Thermo Scientific), in 96-well plates. For analysis all data were fitted by linear regression. IC50 values were determined in 5-fold repeated titration assays.
5
Mitochondrial Superoxide Measurement
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Mitochondrial superoxide levels were measured with MitoSOX red (3,8-phenanthridinediamine, 5-(60-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl) [87 (link),88 (link)] from Molecular Probes (Thermo Fisher Scientific, 29851Willow Creek Road, Eugene, OR, USA). After SH-SY5Y cell treatment in 96-well culture black clear bottom plates, the medium was removed and cells were washed with PBS and afterward, MitoSOX red (final concentration of 5 µM) in 200 µL of PBS was added and incubated for 20 min at 37 °C. After incubation, the cells were washed with warm PBS buffer, and fluorescence was measured at 510 nm excitation/640 nm emission wavelengths in a Varioskan microplate reader (Thermo Fisher Scientific, Vantaa, Finland).
6
Nitric Oxide Production in RAW 264.7 Cells
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Nitric oxide (NO) formation was detected based on the accumulation of nitrites, an indicator of NO synthesis, in the culture medium. RAW 264.7 cells were seeded at 2 × 104 cells/mL in a 96-well plate under SG or HG and supplemented with different concentrations (2.5, 5, 10 and 20 μM) of D3T or RSV and LPS stimulation at 10, 60 or 100 ng/mL for 24 h. After the incubation period, NO concentration was determined by measuring the amount of nitrite produced in the cell culture supernatant using the Griess reagent. The absorbance measurement at 540 nm was performed using a Varioskan microplate reader (Thermo Scientific), and then the results were extrapolated using a calibration curve of sodium nitrite (0 to 1000 µM).
7
Cellular Glutathione Quantification
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Cells were seeded and grown to 80% confluency on 96-well plates. Luminescence based GSH-Glo™ Assay (Promega) was used to determine the cellular glutathione (GSH) levels of 100,000 cells on 96-well according to the manufacturer's manual. The luminescence was measured using a Varioskan microplate reader (Thermo Scientific).
8
Keratinocyte Viability Assay with CNFs
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The cell viability of the human keratinocyte HaCaT cells in the presence of CNFs was determined by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Cells were cultured into a 96-well plate at a density of 5 × 105 cells/well. After 24 h of incubation, the culture medium of each well was replaced with 100 µL of CNFs dispersed in isotonic saline solution at 80 µg/mL, which is the same concentration that was used in the antibacterial tests. This dispersion of CNFs was prepared by sonication during 2 h and used immediately. A positive control of 100 µL of isotonic saline solution without CNFs was also used to replace the culture medium. After 3 h of incubation (higher time exposure than in the antibacterial test), the cells were incubated with 5 mg/mL MTT in each well for 4 h. The formazan was solved in 100 µL dimethyl sulfoxide at 24 ± 1 °C. Finally, absorbance readings were carried out on a Varioskan microplate reader (Thermo Fisher Scientific, Barcelona, Spain) at 550 nm.
9
Cellular Uptake of Hematoporphyrin
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Cellular uptake study of HpD was performed as follow; Cells were incubated for overnight on 12-well plate at 2 × 105 cells/well. The medium was exchanged to flesh one which contained 20 µM of HpD and incubated for 0.5, 1, 3 and 6 h. After incubation, cells were dissolved with 100 µl of self-prepared RIPA buffer.(13 (link)) The cell homogenates were transferred to 96-well plate and then the fluorescence intensity of HpD was measured by a Varioskan micro plate reader (Thermo Fisher Scientific K.K., Kanagawa, Japan). The measurement wavelength of excitation and emission were 415 nm and 625 nm, respectively.
10
Measuring Cellular ROS Production
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Cells (1 × 104 cells/ml) were cultured for 5 days in 96-well plates in DMEM in the presence of glucose and different SCCAs. After washing, cells were incubated with fresh free-serum DMEM (with no glucose or SCCAs), 25 μM dihydroethidium (DHE) was added, and ROS production was monitored every 30 s for 1 h with a Varioskan microplate reader (Thermo Fisher Scientific; Waltham, MA, USA) using λemission of 605 nm and λexcitation of 518 nm.
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