The cytotoxic effect of TQ solution and NPs form was evaluated using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) in the A375 cells. The cells were seeded in 96-well flat plate at a concentration of 1 x 104 cells/well and allowed to attach for 24 h. The cells were then treated with TQ solution, TQ-PLGA NPs and blank NPs using concentration gradients of (1–100 µg/mL) and (0.1–10 mg/mL) respectively for 24 h and 48 h. After the treatment, the medium was aspirated, and the cells were washed with PBS and treated with MTT reagent (5 mg/mL in PBS). The formazan crystals produced by mitochondrial reductase enzyme of the living A375 cells were dissolved using dimethyl sulfoxide (DMSO) with continuous agitation. The cells were incubated at room temperature for 30 min before measuring the light absorbance at 570 nm wavelength using a microplate reader (Infinite M200 Nanoquant, Tecan, Austria). The percentage of cell viability were calculated, by deducting the absorbance of the cell-free wells containing MTT only from the absorbance values of treatment wells and then dividing the corrected absorbance value on by the absorbance of healthy-untreated cells.
Infinite m200 nanoquant
Manufactured by Tecan
Sourced in Switzerland, Austria, United States
The Infinite M200 NanoQuant is a multi-mode microplate reader developed by Tecan. It is designed to perform high-precision absorbance measurements, particularly for nucleic acid and protein quantification. The instrument utilizes a xenon flash lamp and a monochromator-based optical system to provide accurate and reproducible absorbance readings across a wide wavelength range.
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Lab products found in correlation
Whatman, by Cytiva (4 mentions)
Gentra puregene cell and tissue kit, by Qiagen (3 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (2 mentions)
Cell death detection elisa kit, by Roche (2 mentions)
Zr genomic dna tissue miniprep, by Zymo Research (2 mentions)
Rna 6000 nano kit, by Agilent Technologies (2 mentions)
Qubit 3, by Thermo Fisher Scientific (2 mentions)
Fastdna spin kit for soil, by MP Biomedicals (2 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Quantitect rt kit, by Qiagen (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Neutral red assay, by Merck Group (1 mentions)
Oragene collection kit, by DNA Genotek (1 mentions)
Qiaamp dna mini and blood mini kit, by Qiagen (1 mentions)
Qiaamp dna blood mini isolation kit, by Qiagen (1 mentions)
Prepit l2p extraction kit, by DNA Genotek (1 mentions)
Qiaamp dna micro kit, by Qiagen (1 mentions)
Gentra puregene handbook, by Qiagen (1 mentions)
72 protocols using infinite m200 nanoquant
1
Cytotoxicity of Thymoquinone in A375 Cells
2
Equine Pregnancy eCG Levels
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Serum samples of 174 Da’an mare horses with ages between 4 to 11 years were collected three times on day 55, 65, and 75 of pregnancy, respectively. The eCG levels in serum were determined by enzyme-linked immunosorbent assay (ELISA) with commercially available kits (DRG, Marburg, Germany), according to the manufacturer’s instructions. The optical density (OD) at 450 nm was measured using an ELISA microplate reader (infiniteM200 NanoQuant)(TECAN, Hombrechtikon, Switzerland).
3
Genotyping of NNT in Mice
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All mice utilized in this study were genotyped for the wild-type or mutated NNT as described [39] (link). Genomic DNA was isolated from 5 mg of hindbrain as previously described [56] (link). Concentration and purity of DNA was measured at an absorbance of 260 nm and 280 nm on a Tecan infinite M200 Nanoquant (Tecan, Austria).
4
Protein Quantification in CFS-Pa
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The protein concentration of the CFS-Pa was measured using a Bicinchoninic (BCA) Protein Quantification Kit E112 (Vazyme Biotech, Nanjing, China) following the manufacturer’s standard protocol. Initially, the BCA working solution was prepared by mixing reagent A and reagent B at a 50:1 ratio. The standard curve was generated by measuring seven concentrations of BCA working solutions ranging from 0 to 10 μL diluted in water. The BCA working solutions at 0 μL and 10 μL served as blank and reference controls, respectively. For the BCA assay itself, all CFS-Pa samples were diluted 10-fold initially. Then, 10 μL of diluted CFS-Pa samples from each time point was mixed with 100 μL of BCA working solution in a 96-well plate before being mixed briefly. The samples were then incubated at 37 °C for 20–30 min. The absorbance of both the standard curve and CFS-Pa samples was measured at 562 nm (A562) using a UV spectrophotometer (Infinite M200 NanoQuant, TECAN, Männedorf, Switzerland).
5
CRISPR-Chip for Detecting BFP and DMD
6
Genotyping Mice for NNT Mutation
7
Fibroblast RNA Extraction and qRT-PCR
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RNA was isolated from fresh fibroblast cells containing 2 × 106 cells using the RNEasy Plus Mini Kit from Qiagen (cat. no. 74134) following the manufacturer’s instructions. cDNA was synthesized using Qiagen’s Quantitect RT kit (cat no. 205311) following manufacturers recommendations. RNA and cDNA concentrations were determined using the Tecan Infinite M200 Nanoquant plate reader (Tecan, Austria). All primers/probe mix were from Life Technologies (Grand Island, NY, United States). Sequences of commercial primers and probes are proprietary. cDNA was diluted to 40 ng/μl and served as stock template for qRT-PCR. See Supplementary Information and Supplementary Figure 1
8
Evaluating Oleanolic Acid's Impact on Melanoma Cell Growth
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The ability of OA to interfere with melanoma cell growth was evaluated in 501Mel cells in a concentration range of 0.1–200 μM for 48 and 72 h. Cell proliferation was measured using the MTS tetrazolium compound (CellTiter 96 Aqueous One Solution Cell Proliferation assay; Promega, Madison, WI), following the manufacturer’s instructions. Briefly, cells (5 × 103/well) were seeded onto 96-well plates and incubated with various concentrations of compounds for 48 and 72 h in 1% FBS-added medium, to avoid serum proteins interference with compounds. For each treatment, the corresponding vehicle-treated cells were used as control (Ctrl). Final concentration of each solvent in culture medium didn’t exceed 0.2%.
At the end of the treatment, 20 μl MTS solution was added to each well. Absorbance was read at 490 nm using Infinite M200 NanoQuant instrument (Tecan, Salzburg, Austria). Optical density values from vehicle-treated cells were considered as 100% cell viability and the drug concentration giving 50% cell growth inhibition (IC50) was calculated using GraphPad software (GraphPad Prism, version 8.0 from GraphPad Software Inc., San Diego, CA, USA).
At the end of the treatment, 20 μl MTS solution was added to each well. Absorbance was read at 490 nm using Infinite M200 NanoQuant instrument (Tecan, Salzburg, Austria). Optical density values from vehicle-treated cells were considered as 100% cell viability and the drug concentration giving 50% cell growth inhibition (IC50) was calculated using GraphPad software (GraphPad Prism, version 8.0 from GraphPad Software Inc., San Diego, CA, USA).
9
Folin-Ciocalteu Assay for Antioxidant Capacity
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Capability of the extracts and fractions to reduce phosphomolybdic-phosphotungstic acid reagents, otherwise known as Folin–Ciocalteu reagent, was determined according to a previous report with slight modifications [3 (link)]. Briefly, 90 µL diluted Folin–Ciocalteu reagent in deionized water (20% v/v) was placed in each well of a 96-flat-bottomed-well microplate. Then, an 18 µL sample solution in methanol (1000 µg/mL) was added and incubated at room temperature for 5 min, followed by the addition of 90 µL sodium carbonate in deionized water (75 g/L). The mixture was incubated for 2 h at room temperature. The absorbance was then read at 725 nm using a microplate reader (Tecan, Infinite M200 Nanoquant, Männedorf, Switzerland). Gallic acid equivalence (GAE) was determined using a gallic acid calibration curve. All samples were tested in triplicate. Results were expressed as µmol GAE per gram dry weight crude extract or fraction ± standard error of the mean (SEM).
10
Evaluation of mtDNA in Fibroblasts
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