1:2 serial dilutions of ciprofloxacine (200– 0 µg/mL) and sodium hypochlorite (5– 0%) were prepared in Congo Red Broth media with sucrose. S. aureus 29213 were added into culture media with a final concentration of 1×106 Colony-forming unit/mL (CFU/mL). All cultures were performed as triplicate and incubated at 37°C overnight and fluorescence measurements were conducted by using Varioscan (Thermo, USA). The excitation/emission wavelengths were used as 525/625 nm. As a replica plate, bacteria in the same broth media, except Congo Red were cultured and analyzed with conventional Crystal Violet (CV) method. For this purpose, the bacterial cultures were removed, and wells were washed with distilled water. There was 200 µl of 1% CV dye added to each well and incubated at room temperature for 15 min in the dark. Then, CV dye was removed, and wells were washed with water. After the plate was dry, 33% acetic acid was added into wells to dissolve the dye and wells were spectrophotometrically read by Varioscan (Thermo, USA) at 590 nm (Stepanović et al. 2000 (link)).
Varioscan
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Varioscan is a versatile laboratory instrument designed for high-throughput microplate-based assays. It is capable of performing a wide range of photometric measurements, including absorbance, fluorescence, and luminescence detection. The Varioscan offers a flexible and reliable solution for diverse applications in life science research and clinical diagnostics.
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Lab products found in correlation
Transit 2020 reagent, by Mirus Bio (3 mentions)
Passive lysis buffer (plb), by Promega (3 mentions)
Wst 1, by Roche (3 mentions)
Mtt assay, by Merck Group (2 mentions)
Prism 6, by GraphPad (2 mentions)
Ldh cytotoxicity colorimetric assay kit 2, by Abcam (2 mentions)
Wst 1, by Thermo Fisher Scientific (2 mentions)
Hcc1937, by American Type Culture Collection (2 mentions)
Mrc 5, by American Type Culture Collection (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Du145, by American Type Culture Collection (2 mentions)
Micrococcal nuclease, by Takara Bio (1 mentions)
Bsa assay kit, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Total ros rns and superoxide detection fluorescent assay, by Enzo Life Sciences (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Strep tactin hrp, by IBA Lifesciences (1 mentions)
Resazurin, by Thermo Fisher Scientific (1 mentions)
Resazurin, by Merck Group (1 mentions)
46 protocols using varioscan
1
Antimicrobial Susceptibility Screening
2
Fluorescence-based Bacterial Quantification
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The relationship between the number of bacteria and the black color change resulting in fluorescence signal change was investigated. For this purpose, 1:2 serial dilutions (0.5–0 McF) of S. aureus 29213 were prepared in Congo Red Broth media with sucrose. 100 µL of these dilutions were transferred into 96 well plates. All cultures were performed as triplicate and incubated at 37°C overnight and fluorescence measurements were conducted by using Varioscan (Thermo, USA). The excitation/emission wavelengths were used as 525/625 nm. As a replica representing the same conditions, bacteria in MHB media without Congo Red were cultured and spectrophotometrically monitored in real time by Varioscan (Thermo, USA) at 600 nm.
3
Quantitative Autophagy Evaluation in HeLa Cells
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After HeLa cells were treated with compounds, they were washed 2 times with 1X PBS followed by a 10 min incubation with 50 μM MDC in 1X PBS at 37 °C. After 3 washes in PBS, MDC fluorescence intensity was immediately measured with a microplate reader (Varioscan, Thermo Fisher Scientific, US) using excitation/emission maxima 365nm/ 525 nm. Cells were then first treated with 0.02% TritonX-100 in PBS for 10 min and then incubated with 100 μg/ml Propidium Iodide (PI) for 15 min at room temperature. Finally, the plate was read again by multiplate reader (Varioscan, Thermo Fisher Scientific, US) using excitation/emission maxima 535nm/617 nm. After blank subtraction, MDC values were normalized with PI values and the relative MDC specific activity was determined for each sample compared to negative controls (EtOH and DMSO). Fluorometric analysis of MDC was performed in triplicates and quantitative data was presented as mean ± standard error of mean (s.e.m). Data was analyzed by one-way analysis of variance (ANOVA) followed by Turkey's post-hoc test. Statistical analysis was performed using Origin Pro (V8, OriginLab) software.
4
Quantification of Extracellular DNA
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Extracellular DNA released into the culture medium was quantified according to the previously reported protocol (33 (link)). Briefly, the culture medium collected as aforementioned was treated with 1 U/mL micrococcal nuclease (TAKARA Bio Inc. Kusatsu, Shiga, Japan) to partially digest NETs-derived DNA, and was centrifuged at 1,800 × g for 10 min. The DNA fragments recovered in the supernatant were quantified using 1 μM SYTOX Green and a fluorometer (VARIOSCAN, Thermo Fisher).
5
Quantifying RUNX2 Protein Expression
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The OB cells were seeded in gelatin-coated 6-well plates at a density of 200 × 103 cells per well in standard conditions for 96 h. The cells were treated with 150 μL of RIPA Buffer (Sigma Aldrich, St. Louis, MO, USA) and a protease inhibitor cocktail. The total protein level was measured by BSA assay kit (Thermo Scientific, Waltham, MA, USA) using Varioscan (Thermo Scientific, Waltham, MA, USA). An equal amount of protein from each donor was used for the assay. Extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, CA, USA). Primary antibodies for RUNX2 were used for Westernblotting detection of RUNX2 protein.
6
ROS-RNS Production in EA.hy926 Cells
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The total ROS-RNS and superoxide detection fluorescent assay (Enzo Life Sciences, UK) was used to estimate the ROS-RNS production of EA.hy926 cells seeded in a 96-well plate (2×104 cells/well) and treated for 50 hours with NG, IG or HG medium, according to the protocol illustrated in Fig 1 . Pyocyanin was used as a positive control for induction of ROS (200 μM for 20 min) and all treatments were made in triplicate. The assay was performed according to the kit instructions and the fluorescent signal detected (excitation/emission λ = 488/520 nm) using a VarioScan plate reader (Thermo-Fisher) with SkanIt Software 2.4.5 (Varioskan Flash). All results were normalized to the protein content of each well.
7
Cell Viability Assay Protocol
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50,000 cells were plated in a 96-well plate 1 day prior to infections. Three days post-infection cell viability was measured using the The CellTiter-Fluor Cell Viability Assay (Promega), and a multi-well plate reader (Varioscan; ThermoLabsystems) was used to determine the fluorescence of the samples.
8
Hydroxyl Radical Detection via APF Probe Fluorescence
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The probe used for hydroxyl radical measurement was 2-[6-(4′amino phynoxyl-3H-xanthen-3-on-9-yl)] benzoic acid or APF (Thermofisher, Waltham, MA, USA). APF and dimethylformamide (DMF) were mixed in a 1 : 4 vol ratio to make a 1 mM solution of APF in DMF. Next, 10 μL of the APF probe were then mixed with 100 μl of 10× (10 times higher concentration of each of the above samples (in section 2.1). Finally, 890 μl of deionized water was added, yielding an APF probe final concentration of 10 μM). Immediately, each group was irradiated according to the previously presented light parameter and fluorescent intensity (FI) protocols measured at emission and excitation wavelengths 490 and 515 nm using a spectrophotometer (Varioscan®, Thermofisher Scientific, Waltham, MA, USA). Measurements were performed every min from 0 to 5 min and the average FI was calculated. Ten μM of a standard curcumin solution and probe served only as positive and negative controls, respectively.
9
Enzyme Kinetics of Cb FDH Mutants
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Activity assays were performed for wild-type and mutant CbFDH at different substrate, coenzyme and enzyme concentrations in order to calculate the Km, Vmax and kcat values. The values were used to plot Michaelis–Menten and Lineweaver–Burk graphs, which yielded an equation for calculations. The reaction conditions used for measurements are shown in Table 1 ▸ .
The measurements were performed at 25°C and 340 nm in a microplate reader (Varioscan, ThermoFisher Scientific, USA).
The measurements were performed at 25°C and 340 nm in a microplate reader (Varioscan, ThermoFisher Scientific, USA).
10
Evaluating Cell Viability in A549 Cells
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Human lung adenocarcinoma epithelial cell line A549 cells were cultured at 37 °C in growth medium (DMEM with 10% fetal bovine serum) in 5% CO2, and then seeded into 96-well plates at a density of 1 × 105 cells/mL. Once the cells reached 80%–90% confluence, they were treated with or without 10 µM of various compounds at 37 °C for 12 h. Next, 10 μL Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) solution was added to each well, and the plate was incubated for 2 h at 37 °C. Cell viability was determined by measuring the absorbance at 450 nm using a fluorimeter (Varioscan, Thermo, USA).
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