The cells were seeded at a density of 1.5 × 104 cells/ml of medium in a 96-well plate (0.2 ml/well). The cells were incubated for 24 h before treatment with DOC and/or ROB for 72 h. Following treatment, 10 µL Cell Counting Kit-8 (CCK-8) (CK04-11, VitaScientific, Beltsville, MD, USA) was added into each well and the cells were incubated for 30 min. A microplate reader ChroMate™ (Awareness Technology, Palm City, FL, USA) was used to detect the optical density (OD) at a wavelength of 450 nm.
Chromate
Manufactured by Awareness Technology
Sourced in United States
The ChroMate is a compact, automated microplate reader designed for a variety of spectrophotometric analyses. It features a high-performance monochromator that enables precise wavelength selection across a wide range, allowing for absorbance, fluorescence, and luminescence measurements. The ChroMate provides reliable and reproducible results, making it a versatile tool for diverse applications in research and clinical laboratories.
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8 protocols using chromate
1
Cell Viability Assay with DOC and ROB
2
hFOB Cell Adhesion on PLA/ZrO2 Nanocomposite
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As the first attempt to investigate how hFOB cells would interact with the PLA/ZrO2 nanocomposite fiber material, adhesion assays were performed. For this purpose, hFOB 1.19 cells were seeded at 1 × 104 cells/mL onto the composite scaffold in triplicate and were allowed to adhere under standard cell culture conditions for 4 and 24 h. After each time, nanocomposite fiber scaffolds were rinsed three times using phosphate-buffered saline (PBS) to remove non-adherent cells. Next, the adherent cells were fixed with 3.5% paraformaldehyde, and the evaluation of cell attachment was performed according to the crystal violet assay. Briefly, cells were incubated with 0.1% crystal violet solution for 15 min and then washed carefully with deionized water until clear water was obtained. Then, the dye was extracted with 0.1% of sodium dodecyl sulfate (SDS), and the optical absorption was quantified by spectrophotometry at 550 nm with an ELISA plate reader (ChroMate, Awareness Technology). Cells attached in triplicate onto a conventional tissue culture plate (TCP) were considered as 100% cell attachment of hFBO 1.19 cells, and cells seeded in triplicate onto PLA fiber membrane scaffolds were used as the control.
3
ELISA-based Soybean Mosaic Virus Detection
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The leaf samples were collected from the uppermost trifoliolate leaves of the plants. These samples were assayed using an ELISA complete kit (BIOREBA, Switzerland) according to the manufacturer’s protocol. The SMV levels were determined by measuring the absorbance of the samples at a wavelength of 405 nm (OD405) using an ELIZA reader (ChroMate®, Awareness Technology, Romer Labs, Inc., USA). A sample was considered positive for SMV if the OD405 reading was three or more times greater than that of a healthy plant extract. The test was performed between 30 and 120 min after substrate addition [23 (link)].
4
MTS Assay for Cell Proliferation
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To evaluate cell proliferative capacity, we used CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega), based on tetrazolium compound (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium; MTS). Cells were seeded in a 96-well iMatrix-511-coated plate after Y-27632 treatment or no treatment. The reagent was added to each sample each day. After a 2-h incubation at 37 °C, absorbance was measured at 492 nm in a microplate reader (ChroMate, Awareness Technology).
5
Cytotoxicity Evaluation of Amorphous SPs
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The cytotoxicity of amorphous SPs was determined using a WST-1 assay (Takara, Tokyo, Japan). This is a method to evaluate viable cells by enzymatic cleavage of the tetrazolium salt WST-1 to formazan by mitochondrial dehydrogenases expressed by viable cells. Balb/3T3 was seeded at 2×104 cells/well (48-well plate) in the culture medium. After incubation for 24 h, the culture supernatant was removed, and the SPs or an equal volume of HBSS was treated in culture media (the final concentration of FCS was around 5%). The SP treatment conditions were 11, 33.3, 100, and 300 µg/mL for 24 h. WST-1 mixtures were added to each well and incubated for 2 h at 37 °C in a humidified atmosphere of 5% CO2 in air. After incubation, 100 µL of the cell culture supernatant, including WST formazan, was transferred to a 96-well plate, and the absorbance was measured using a microplate reader (ChroMate®, Awareness Technology Inc. Florida, USA) at a wavelength of 450 nm.
6
Hemolytic Activity of Peptides
7
Nitric Oxide Colorimetric Assay
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The level of nitrates was assayed in the samples using a nitric oxide assay kit (AB65328, Abcam, Cambridge, MA, USA) at 540 nm in a colorimetric microplate reader ChroMate® microplate reader (Awareness Technology, Palm City, FL, USA). In brief, cells (1 × 10 6 ) were treatment with or without AGP for the indicated time points, then harvested and washed the cells with cold PBS.
Resuspended and homogenized cells in 100 µL of ice cold assay buffer, centrifuge cell for 3 min at 4°C at 13,000 × g. Added ice cold 4M perchloric acid (PCA) to a final concentration of 1 M in the homogenate solution, then centrifuged samples at 13,000 × g for 2 min at 4°C. Precipitated excess PCA by adding an equal volume of cold 2 M KOH to supernatant (adjust pH to 6.5-8). Centrifuge at 13,000 × g for 15 min at 4°C and the supernatant was used in the assay.
Resuspended and homogenized cells in 100 µL of ice cold assay buffer, centrifuge cell for 3 min at 4°C at 13,000 × g. Added ice cold 4M perchloric acid (PCA) to a final concentration of 1 M in the homogenate solution, then centrifuged samples at 13,000 × g for 2 min at 4°C. Precipitated excess PCA by adding an equal volume of cold 2 M KOH to supernatant (adjust pH to 6.5-8). Centrifuge at 13,000 × g for 15 min at 4°C and the supernatant was used in the assay.
8
LDH Cytotoxicity Assay Protocol
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Cell culture media was assessed for lactate dehydrogenase (LDH) 24 h after treatment using the Cytotox 96 assay (Promega) according to the manufacturer’s instructions. Absorbance was measured using a ChroMate microplate reader (Awareness Technology Inc., Palm City, FL).
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