crystal violet was used for cell viability quantification. The culture medium was removed, and cells were gently washed with PBS, fixed with 1% glutaraldehyde (Merck) in PBS for 15 min and stained with 0.1% crystal violet (Sigma) in water for 30 min. The plates were washed under running tap water and subsequently lysed with 0.2% Triton X-100 (Roche) in water for 90 min. The associated absorbance was measured at 590 nm using the BioTek® 800™ TS Absorbance Reader.
Biotek 800 ts absorbance reader
Manufactured by Agilent Technologies
Sourced in United States
The BioTek-800-TS is an absorbance reader designed for use in life science laboratories. It is capable of measuring the absorbance of samples within a specified wavelength range, providing quantitative data for various analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Microlab 300, by ELITechGroup (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Triton x 100, by Roche (1 mentions)
Enzychrom glutamine assay kit, by BioAssay Systems (1 mentions)
Sorafenib, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Thermo Fisher Scientific (1 mentions)
Mitomycin c, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Prism 8, by GraphPad (1 mentions)
Doxorubicin, by Pfizer (1 mentions)
Sorafenib, by Bayer (1 mentions)
Glutamine assay kit, by BioAssay Systems (1 mentions)
α kg colorimetric assay kit, by Abcam (1 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Dimethyl sulfoxide (dmso), by Keygen Biotech (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Keygen Biotech (1 mentions)
High sensitivity salivary cortisol enzyme immunoassay kit, by Salimetrics (1 mentions)
Cell proliferation kit 1, by Roche (1 mentions)
17 protocols using biotek 800 ts absorbance reader
1
Cell Viability Quantification via Crystal Violet
2
Quantifying Cellular Glutamine Consumption
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To evaluate the glutamine consumption of cells, the level of glutamine in the culture medium was measured with the EnzyChromTM Glutamine Assay Kit (Bioassay Systems, Hayward, CA, USA), according to the manufacturer’s instructions. Briefly, the cell medium was harvested and mixed with the enzyme after deproteinization with an ultracentrifugal filter. The absorbance at 565 nm was measured with a BioTek™ 800TS Absorbance Reader (BioTek) after incubation for 40 minutes in the dark and the concentration of glutamine was calculated by the given kit formula. Then, the glutamine consumption was calculated according to the following equation: Glutamine consumption = (the concentration of glutamine in fresh medium − the concentration of glutamine in cell medium) × the volume of cell medium. The final amount of glutamine consumption was quantified to the content of whole cellular protein and the result was determined as μmol glutamine/mg protein.
3
Chemosensitivity of HCC Cell Lines
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The sensitivity of Oncopig, human, and murine HCC cell lines was tested for five chemotherapeutic agents: sorafenib (Bayer, Leverkusen Germany), doxorubicin (Pfizer Inc., New York NY, USA), cisplatin (Sigma-Aldrich, St. Louis MO, USA), mitomycin C (Accord Healthcare Inc., Durham NC, USA), and 5-FU (Acros Organics, Geel Belgium). Briefly, 1 × 104 cells/well (2 × 104 for HepG2) were seeded in 96-well plates. The following day, culture medium was replaced with fresh medium supplemented with each chemotherapeutic agent at 8-point serial dilutions. The following drug concentrations were used to assay clinically relevant administered dosages [34 (link), 35 (link)], and to allow calculation of the IC50: sorafenib and mitomycin C: 0.5–100 μM, doxorubicin: 0.1–20 μM, cisplatin: 1–200 μM, and 5-FU: 1–500 μM. Cell viability was assessed after 72 hours using a MTT assay (#V13154; Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendations using a BioTek 800 TS Absorbance Reader (BioTek, Winooski, VT, USA). IC50 values were determined by nonlinear regression analysis using GraphPad Prism 8 (GraphPad, San Diego, CA, USA) from plots of relative percent viability versus log10 drug concentration.
4
Glutamine Consumption and Metabolite Levels
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The level of glutamine in the culture medium was measured with a Glutamine Assay Kit (Bioassay Systems, Hayward, CA, USA). Briefly, the cell medium was mixed with the enzyme after deproteinization with an ultracentrifugal filter. The absorbance at 565 nm was measured with a BioTek™ 800TS Absorbance Reader (BioTek) after incubation for 40 min in the dark. Glutamine consumption was calculated according to the following equation: Glutamine consumption = glutamine in fresh medium-glutamine in cell medium.
The levels of α-KG and ATP in cultured cells were assessed with α-KG colorimetric assay kit (BioVision, Milpitas, CA, USA) or CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) in accordance with manufacturer’s instructions.
The levels of α-KG and ATP in cultured cells were assessed with α-KG colorimetric assay kit (BioVision, Milpitas, CA, USA) or CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) in accordance with manufacturer’s instructions.
5
Fibroblast Viability on ZnO-NP Membranes
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Adult human dermal fibroblasts were seeded on electrospun membranes with different concentrations of the ZnO-NPs (0%, 0.5%, 1%, 2%, 3%, and 4%). After sterilization with ethanol 70%, 5 × 10 3 cells were seeded on each sample in 96-well plates and incubated at 37°C with 94% humidity and 5% CO 2 . Cell viability and proliferation were monitored at days 1, 3 and 7 using MTT assay. Dulbecco's modified Eagle's medium (DMEM) (Gibco, Waltham, USA; Mfr. No. Gibco ™ 31600083) enriched with 10% of fetal bovine serum (FBS) (Gibco, Mfr. No. Gibco ™ 10082139) and 1% penicillin/streptomycin (Gibco, Mfr. No. Gibco ™ 15140122) was used as culture medium. At each time point, 20 µL of MTT solution (Sigma-Aldrich; CAS No. 298-93-1) was added to each well. Cell culture plates were incubated for 3 h; then, the media was replaced with 200 µL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich; CAS No. 67-68-5), and the cultures were incubated for another 30 min. 14, 15 Finally, the absorbance of each sample was determined using a BioTek 800 ™ TS Absorbance Reader (BioTek Instruments Inc., Winooski, USA) at 570 nm. Each sample was evaluated in triplicate.
6
Evaluating Cisplatin Sensitivity in Ovarian Cancer Cells
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A2780/DDP and SKOV3/DDP cells were exposed to 0, 2.5, 5, 10, 20, 40, or 80 μM DDP for 48 h. A2780/CP and SKOV3/CP cells were exposed to 0, 1, 10, 100, or 1000 μM CP for 48 h. A2780/DDP cells with stable knockdown of SH3RF2 were transiently transfected with RBPMS siRNA for 24 h and then treated with 30 μM DDP for 48 h. The MTT assays were performed according to manufacturer’s instructions. In brief, the MTT (50 μL; KeyGEN, China) was added to each well. After 4-h incubation with MTT, the supernatants of cell cultures were removed and then dimethyl sulfoxide (DMSO; 150 μL; KeyGEN) was added. The optical density (OD) values were measured at 490 nm by the BioTek® 800™ TS Absorbance Reader (BioTeK, USA).
7
Saliva Cortisol Sampling and Quantification
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To collect saliva, participants were asked to keep oral swabs under the tongue for 2 min. Immediately after collection, saliva samples were frozen at − 20 °C. For the analysis, samples were thawed, brought to room temperature and centrifuged (1500 g × 10 min), resulting in a clear supernatant of low viscosity. Salivary cortisol levels were determined by enzyme-linked immunosorbent assay (High Sensitivity Salivary Cortisol Enzyme Immunoassay Kit: Salimetrics LLC, State College, PA) by a researcher who was blind to the design of the experiment. Samples were assayed in duplicates following kit instructions with a 96-well plate, using the BioTek 800 TS absorbance reader and Gen5 software (BioTek Instruments Inc., Vermont, USA). To avoid inter-assay variability all samples from the same participant were assayed in the same batch. The inter-assay and intra-assay coefficients of variability were 5.9 and 8.4, respectively. Specific analyses are detailed below.
8
MTT Assay for Cell Viability Assessment
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The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was conducted using the Cell Proliferation Kit I (Roche) to assess cell viability. In short, ESCC cells were transfected with oligonucleotides and/or plasmids and then incubated onto 96-well plates for 48 h. Thereafter, the MTT reagent (5 mg/mL, 10 μL) was added to each well and incubated for 4 h. Subsequently, the solubilization solution (100 μL) was supplemented to dissolve the purple crystals. The OD (optical density) value of the solution at 570 nm was measured with a BioTek™ 800TS Absorbance Reader (BioTek, Winooski, VT, USA).
9
Determination of Total Antioxidant Capacity
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Total antioxidant capacity estimated as gallic acid equivalent (GAE) was determined as earlier described by Urbano et al. [18 ]. A final mixture containing 1 ml of ABE and 3 ml of a solution made up of sulphuric acid, disodium phosphate, and ammonium molybdate at concentrations 0.6 M, 28 mM, and 4 mM, respectively, was incubated (Yohmai IN-601, Stains, France) at 95°C for 90 min. The absorbance of the mixture was determined at 695 nm (BioTek-800-TS absorbance reader, Agilent, Santa Clara, USA) upon cooling. Gallic acid was used to establish a standard curve with the GAE of ABE being extrapolated from the obtained curve.
10
Quantifying SARS-CoV-2 Spike Protein Binding to NRP-1
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All reagents and substances were brought to room temperature (18–25 °C) before use. The RayBio COVID-19 Spike-NRP-1 Binding Assay Kit I contains a 96-well plate coated with recombinant NRP-1. Serial dilutions of folic acid, leucovorin, and lopinavir were added into the wells in the presence of recombinant spike S1 protein, according to the manufacturer’s instructions. Unbound S1 was removed with a wash step, and a mouse anti-S1 IgG detection antibody was added in order to bind to the S-NRP-1 complex. After washing, an HRP-conjugated anti-mouse secondary IgG was then applied to the wells in the presence of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The HRP reacted with the TMB solution, producing a blue color that was proportional to the amount of bound S1. The HRPTMB reaction was stopped with the addition of the Stop Solution, resulting in a blue-to-yellow color change. The intensity of the color was then measured at 450 nm (BioTek 800 TS Absorbance Reader, Agilent, Santa Clara, CA, USA).
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