The viability of cultured cells was determined by assaying the reduction of 3-(4,5-dimethyl thiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) to formazan [25 (link)]. Briefly, PC12 cells were cultured in a 96-well microliter plate at a density of 5000 cell/well. After pretreatment with rutin (0.5, 1, 1.5, 2.5, 5, 10, 20, 40, 80 and 160 μM/ml) for 24 h, ACR at concentration of 5.46 mM was added to each well. The cells were then incubated for 48 h and then treated with MTT solution (0.5 mg/ml PBS) for 1 h at 37°C. Upper mediate replaced with dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm (630 nm as reference) in a plate reader (TECAN infinit M200) [14 ].
Infinit m200
Manufactured by Tecan
Sourced in Switzerland
The Tecan Infinite M200 is a multi-mode microplate reader designed for diverse application requirements. It offers wavelength ranges from 230 to 850 nm and supports a variety of detection modes including absorbance, fluorescence, and luminescence. The instrument is capable of handling 6- to 384-well microplates and can perform temperature-controlled measurements.
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Lab products found in correlation
6 protocols using infinit m200
1
MTT Assay for Evaluating Cell Viability
2
Liposome Cytotoxicity Evaluation in Cells
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The cytotoxicity of liposomes was determined by the MTT assay in MDCK and DC2.4 cells. Cells were seeded in DMEM or RPMI1640 supplemented with 10% FBS at a density of 2 × 105 cells/mL in a 96-well plate and cultured overnight. Cells were treated with liposomes (25, 50, 100, or 200 μg/mL) in the presence or absence of dLOS (0.5, 1, 2, or 4 μg/mL) for 24 h. The next day, CCK-8 solution (Dojindo, Tokyo, Japan) was added to the culture, and cells were incubated for 2 h. Optical density at 450 nm was measured using a microplate reader (Infinit M200; Tecan, Mannedorf, Switzerland). All assays were performed in triplicate. Cell viability relative to that of control cells was calculated as: cell viability (%) = ((OD450 of test cells − OD450 of blank well)/(OD450 of media control cells − OD450 of blank well)) × 100.
3
Quantifying IAA Production by Bacterial Isolates
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To determine amounts of IAA produced by each isolate, 1 mL of bacterial culture in ISP2 broth was inoculated in Tryptic Soy Broth supplemented with 150 mg/L L-tryptophan. Approximately 1 mL of culture solution was centrifuged at 12000 rpm for 5 min, and 1mL of the supernatant was mixed with 2 mL of Salkowski’s reagent (150 mL concentrated H2SO4, 250 mL distilled water, 7.5 mL 0.5 M FeCl3.6H2O) and incubated for 20 min in darkness at room temperature (de Oliveira-Longatti et al., 2014 (link)). IAA production was qualitatively assayed as pink color development and quantified by measurement of absorbance at 530 nm using a spectrophotometer infinit M200 (Tecan, Switzerland). The calibration plot was constructed using dilutions of a standard IAA (Fluka, Switzerland) solution and the uninoculated medium with the reagent as a standard curve (0, 5, 10, 20, 50, and 100 μg/mL). The quantity of IAA in the culture was expressed as μg/mL.
4
Quantitative Analysis of Succinic Acid and 2-OG in LV Samples
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The quantitative measurements of succinic acid and 2-OG in LV samples were obtained from metabolic analysis data at the Human Metabolome Technologies (HTM, Tsuruoka, Japan), using LV samples from four normal and six CHF dogs. The quantitative measurement of succinic acid in HCF was performed using a Succinate Assay kit (Colorimetric) (ab204718, Abcam) following the manufacturer’s instructions. Colorimetry of succinic acid was determined at 450 nm, using an InfinitM200 (TECAN, Mannendorf, Switzerland). Quantification analyses were performed in triplicate.
5
PAMPA Assay for Blood-Brain Barrier
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The parallel artificial membrane permeability assay (PAMPA) blood–brain barrier (BBB) experiments were conducted using the Pampa Explorer Kit (Pion Inc., Billerica, MA, USA) according to the protocol provided by the manufacturer. Briefly, the donecopride fumarate solution (20 mM in DMSO) was diluted in Prisma HT buffer (pH 7.4; pION) to 100 μM; 200 μL this solution (n = 6) was added to the donor plate (P/N 110,243). Five microliters BBB-1 Lipid (P/N 110,672) was used to coat the membrane filter of the acceptor plate (P/N 110,243). Two hundred microliters Brain Sink Buffer (P/N 110,674) was added to each well of the acceptor plate. The PAMPA sandwich was assembled and allowed to incubate at room temperature for 4 h without stirring. The sandwich was then separated, and the UV-visible spectra were measured for both the donor and receiver wells with the microplate reader (Tecan Infinit M200). The −logPe and Pe (PAMPA effective permeability coefficient) were calculated by the PAMPA Explorer software version 3.7 (pION) for studied compounds. Corticosterone (−logPe = 4.76, Pe = 17.2 × 10−6 cm/s) and theophylline (−log Pe = 6.44, Pe = 0.40 × 10−6 cm/s) were used as positive and negative references, respectively.
6
Lipid and Carbohydrate Analysis in Cells
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Observation and measurements of lipids in cells were performed by Oil Red O staining using a Lipid Assay kit (Cosmo Bio, Tokyo, Japan). Carbohydrates were observed by PAS staining24 (link) and their levels were measured using the phenol-sulfuric acid method with a Total Carbohydrate Colorimetric Assay kit (BioVision, Milpitas, CA). Colorimetry of lipids and carbohydrates was carried out at 540 and 490 nm, respectively, using an InfinitM200 (TECAN, Mannendorf, Switzerland).
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