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Infinite m200

Manufactured by Tecan
Sourced in Switzerland, Austria, United States, Germany, Japan, China, United Kingdom, Belgium, Sweden, Australia, Spain, France

The Infinite M200 is a multi-mode microplate reader that provides high-performance absorbance, fluorescence, and luminescence detection. It offers precise and versatile measurement capabilities for a wide range of applications in life science research and drug discovery.

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2 648 protocols using infinite m200

1

Cell Viability and LDH Assay Protocol

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Cell viability was determined with Cell Counting Kit‐8 (CCK‐8). A total of 2 × 103 cells/wells were seeded in a 96‐well plate after 48 h of transfection. Cell viability was detected using an ELISA reader (Infinite® M200, Tecan trading AG) at 450 nm.
Then, LDH activity levels were determined using the LDH activity kit. A total of 2 × 103 cells/wells were seeded in a 96‐well plate after 48 h of transfection. LDH activity levels were detected using an ELISA reader (Infinite® M200, Tecan trading AG) at 450 nm.
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2

Quantification of NOX and XO Activities

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NADPH oxidase (NOX-EC 1.6.3.1) activity was examined by the luminescent method using lucigenin as a luminophore [91 (link)]. Cell lysates were added to phosphate buffer (50 mM, room temperature, pH 7.0) supplemented with ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (1 mM, EGTA), saccharose (150 mM), NADPH (10 mM) and lucigenin (1 mM). The luminescence was measured for one minute immediately after mixing the sample (Microplate reader Infinite M200, TECAN Trading AG, Männedorf, Switzerland). The amount of enzyme required to release 1 nmol of superoxide anion radicals per minute was equivalent to one unit of NOX activity and is expressed in relative luminescence units (RLU). Enzyme-specific activity is shown in relative luminescence units (RLU) per mg of protein.
Xanthine oxidase (XO-EC1.17.3.2) activity was estimated by uric acid formation from xanthine [92 (link)]. Cell lysates were added to phosphate buffer (50 mM, room temperature, pH 7.5) supplemented with xanthine (150 µM). Next, the changes in absorbance at 290 nm were measured for one minute immediately after mixing the sample (Microplate reader Infinite M200, TECAN Trading AG, Männedorf, Switzerland). One XO unit was defined as the amount of enzyme required to release 1 µM uric acid per minute. Enzyme activity is shown in microunits per mg of protein.
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3

Enzymatic Assays for Liver Health

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Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) from samples of 20 μl serum or tissue homogenate were determined according to modified methods described by Casillas et al. (1982) after incubation for 30 min at room temperature by monitoring NADH oxidation at 339 nm (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). Sorbitol dehydrogenase (SDH) activity, that is responsible for the reversible conversion of fructose to sorbitol and vice versa, was measured in serum samples as described by Bergmeyer (1974b) using fructose as substrate. Absorption changes at a wavelength of 339 nm were recorded for 10 min (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland).
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4

Transient Transfection and Luciferase Assay

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PC‐3 cells were transiently transfected with different vectors along with the reporter vector containing luciferase gene (Luc) or full‐length cyclin A1 promoter in Luc reporter vector (cyclin A1‐Luc) as indicated. The Firefly Luciferase and Renilla Luciferase activity were determined by using an Infinite® M200 multimode microplate reader (Tecan Sunrise™), equipped with dual injector. For AR receptor activity assay in LNCaP cells, AR Cignal Reporter Assay Kit (Qiagen Inc.) was used according to the manufacturer’s protocol. Briefly, LNCAP cells were transiently transfected with different vectors along with the reporter vector ARE‐luciferase (ARE‐Luc) vector as indicated. The Firefly and Renilla Luciferase with Dual Luciferase Assay Kit (Promega, Biotech Sweden, Stockholm, Sweden) according to standard protocol in the Tecan Infinite M200 (Tecan Trading AG, Mannedorf, Switzerland) plate reader equipped with dual injector.
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5

Evaluating Cytotoxicity of Anti-Cancer Compounds

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To determine the effects of 5-FU/PTX, CLZ and each CPP2–thiazole derivate on the viability of HT-29 and PC-3 cells, MTT and SRB assays were used. For the MTT protocol, after drug treatment, the cell medium was removed and 100 µL/well of MTT solution (0.5 mg/mL in PBS) was added. Cells were incubated for 3 h, protected from light. After this period, the MTT solution was removed, and DMSO (100 µL/well) was added to solubilise the formazan crystals. Absorbance was measured at 570 nm in an automated microplate reader (Tecan Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). For the SRB assay, after treatments, the cultured cells were fixed with ice-cold 10% trichloroacetic acid for 30 min and stained with 0.4% SRB for 1 h at room temperature. Excess dye was removed by rinsing several times with tap water. Protein-bound dye was dissolved with 200 µL 10 mM Tris base solution for the determination of absorbance with a microplate reader with a filter wavelength of 540 nm (Tecan Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). The IV of the therapeutic drug was determined as each drug concentration showing 50% cell growth inhibition compared with the control. All conditions were performed three times independently in triplicate.
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6

Cytotoxicity Evaluation of Drugs

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Two cell-based assays were used to determine the antitumor effect of each drug alone and in combination on MCF-7 cells: MTT and SRB assays. For the MTT assay, after cell seeding and treatment, cell media was aspirated and replaced with 100 μL per well of MTT solution (0.5 mg/mL in phosphate-buffered saline (PBS)). Then, cells were maintained at 37 °C for 3 h in a light-protected manner. At the end of this time, the MTT solution was aspirated, and 100 μL/well of DMSO was added to dissolve the formazan crystals. The measure of absorbance at 570 nm was performed using an automated microplate reader (Tecan Infinite M200, Tecan Group Ltd, Männedorf, Switzerland).
Regarding the SRB protocol, after cell seeding and treatment, cells were fixed using an ice-cold 10% trichloroacetic acid solution for 30 min and incubated with a 0.4% SRB solution for 1 h at room temperature. To remove the excess dye, plates were washed three times with tap water and allowed to dry. To quantify protein-bound dye, 200 µL of 10 mM Tris base solution was added to each well, and absorbance readings were performed at 510 nm using an automated microplate reader (Tecan Infinite M200, Tecan Group Ltd, Männedorf, Switzerland). Each experiment was repeated three times, using triplicates.
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7

Cytotoxicity Assays for Cell Lines

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To determine the effects of 5-FU or PTX and the repurposed drugs on the viability of HT-29 and MCF-7 cells, respectively, MTT and SRB assays were used. For the MTT protocol, after drug treatment, the cell medium was removed and 100 µL/well of MTT solution (0.5 mg/mL in PBS) was added. Cells were incubated for 3 h, protected from light. After this period, the MTT solution was removed, and DMSO (100 µL/well) was added to solubilise the formazan crystals. Absorbance was measured at 570 nm in an automated microplate reader (Tecan Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). For SRB assay, after treatments, the cultured cells were fixed with ice-cold 10% trichloroacetic acid for 30 min and stained with 0.4% SRB for 1 h at room temperature. Excess dye was removed by rinsing several times with tap water. Protein-bound dye was dissolved with 200 µL 10 mM Tris base solution for the determination of absorbance with a microplate reader with a filter wavelength of 540 nm (Tecan Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). The IC50 of the therapeutic drug was determined as each drug concentration showing 50% cell growth inhibition as compared with control. All conditions were performed three times independently, in triplicate.
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8

Measuring Mitochondrial and Plasma Membrane Potentials

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Mitochondrial membrane potential was determined using tetramethylrhodamine, ethyl ester (TMRE from ThermoFisher). 1 mM TMRE was diluted into cell culture medium with indicated treatments to a final concentration of 200 nM. Hoechst was added to a final concentration of 10 μg/mL. Cells were incubated with TMRE/Hoechst for 30 minutes and washed two times with Hank’s Balanced Buffer Solution (HBSS with Ca2+ and Mg2+). Cells were imaged using a Cytation 1 Cell Imaging MultiMode Reader from BioTek. TMRE intensity was normalized to total cell counts for each image.
Plasma membrane potential was determined using Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol (DiBAC4(3)) (ThermoFisher). 1 mM DiBAC4(3) was diluted into cell culture medium with indicated treatments to a final concentration of 200 nM. Hoechst was added to a final concentration of 10 μg/mL. Cells were incubated with DiBAC4(3)/Hoechst for 30 minutes and washed two times with Hank’s Balanced Buffer Solution (HBSS with Ca2+ and Mg2+). Fluorescence intensity was determined using a Tecan infinite M200 plate reader. DiBAC4(3) intensity was normalized to total cell counts for each image.
For correlation of mitochondrial and plasma membrane potential both dyes were used simultaneously with Hoechst. Fluorescence intensity was then determined using a Tecan infinite M200 plate reader.
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9

Oxidative Stress Biomarkers in Plasma and Liver

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Antioxidant capacity of plasma: The antioxidant capacity of plasma was evaluated by the oxygen radical absorbance capacity (ORAC) method, as previously described by Manso et al. [16 (link)], and measured fluorometrically (485 nm excitation and 520 emission; FLUOstar Optima, BMG Labtech GmbH, Offenburg, Germany). The results were expressed as µmol of trolox (Sigma, St. Louis, MO, USA) equivalent/µL of plasma.
Plasma malondialdhehyde: Plasma concentration of malondialdehyde (MDA) was determined by the thiobarbituric acid assay and spectrophotometrically measured at 535 nm (Infinite M200, Tecan, Switzerland) as previously reported by Manso et al. [16 (link)]. The results were expressed as nmol MDA/ml plasma.
Glutathione Reduced in liver: The levels of reduced glutathione (GSH) were measured by the monochlorobimane fluorimetric assay, previously reported by Kamencic et al., by a fluorometer (390 nm excitation and 510 emission; Infinite M200, Tecan, Switzerland) [17 (link)]. The results were expressed as µmol GSH/g protein.
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10

Superoxide and Hydrogen Peroxide Detection

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Superoxide detection was performed using L-012 in spheroid cultures in the presence of 2.5 μg/mL LPS, 2.5 μg/mL Poly(I:C) HMW (Invivogen), 20 μM ML171 (Tocris), 1 mM butyrate (Sigma) or 5 μM Gefitinib (Cayman). Cells in matrigel were recovered in prewarmed 37°C Krebb-ringer bicarbonate buffer containing 100 μM L-012 (FUJIFILM, Wako) and transferred to white 96-well plate. Luminescence was measured over a 30 minutes time window at 37°C using an Infinite m200 (Tecan) or CLARIOstar (BMG Labtech) plate reader. Spheroids were isolated from the matrigel using recovery solution as described before and BCA was used to quantify the protein concentration used to normalize the assay results. H2O2 generation was measured using the Amplex red assay (Thermo Fisher). 100μL of 0.2U/mL HRP, 20μM Amplex red in preheated 37°C Krebs-ringer bicarbonate buffer was added to each condition and incubated for 1 hr. at 37°C. The reaction was quenched and spheroids were disrupted by adding 20 μL 5 mM EDTA and the supernatant was transferred to a 96 well plate. In parallel a standard H2O2 curve was made prepared to determine the absolute nmol concentration in the samples. Analyzes were performed on using an Infinite m200 plate reader (Tecan) at excitation 530 nm and emission 600 nm.
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