U2OS, A2780 and MDA-MB-231 cells were purchased from ATCC and were maintained in McCoy’s 5A or RPMI 1640, respectively, supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic–antimycotic (Gibco) and incubated at 37 °C with 5% CO2. Cell viability assays were carried out by plating 2,000 cells per well in 96-well plates. Cells were treated with the relevant drug for 24 h or 72 h, then incubated with CellTiter-Blue (20 μl/well) for 1 h before recording fluorescence (560(20)Ex/590(10)Em) using a PerkinElmer Wallac 1420 Victor2 (link) Microplate Reader. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol, stained with propidium iodide (10 mg ml–1 PI, 250 mg ml–1 RNase A, 0.5% BSA, 0.02% sodium azide in 1× PBS) and analysed by FACS on a CyFlow Ploidy Analyser from Partec.
Wallac 1420 victor2
Manufactured by PerkinElmer
Sourced in United States, Italy, Finland
The Wallac 1420 Victor2 is a multi-mode microplate reader designed for a wide range of applications in life science research. It is capable of performing various detection modes, including absorbance, fluorescence, and luminescence measurements. The instrument is equipped with a temperature-controlled incubator and shaker, allowing for precise control of experimental conditions.
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Lab products found in correlation
One glo luciferase assay substrate, by Promega (6 mentions)
Celltiter glo luminescent cell viability assay, by Promega (4 mentions)
Prestoblue, by Thermo Fisher Scientific (3 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Cell proliferation kit 1, by Roche (2 mentions)
Luciferase assay kit, by Promega (2 mentions)
Opti mem media, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx plus, by Thermo Fisher Scientific (2 mentions)
Nf κb luciferase reporter, by Thermo Fisher Scientific (2 mentions)
Caspase glo 3 7 assay kit, by Promega (2 mentions)
Antibiotic antimycotic, by American Type Culture Collection (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Fluo 8, by PerkinElmer (1 mentions)
Fluo 8 am, by Abcam (1 mentions)
Dcf da, by Thermo Fisher Scientific (1 mentions)
Ethidium bromide, by Thermo Fisher Scientific (1 mentions)
90 protocols using wallac 1420 victor2
1
Cell Viability and Cell Cycle Assays
2
Matriptase Proteolytic Activity Assay
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The synthetic, fluorogenic substrate (N-t-Boc)-Gln-Ala-Arg-AMC was used to assess matriptase proteolytic activity. The reaction buffer with a final volume of 100 μl contained 20 mM Tris–HCl, pH 8.5 and the fluorogenic substrate at a final concentration of 10 μM. The hydrolysis of the peptide substrate was recorded every minute for a total of 30 or 40 min using a Wallac 1420 Victor 2 (PerkinElmer®) Microplate Reader, using an excitation wavelength of 355 nm and measuring emission at 460 nm.
3
Cell Viability Assay for Compound Screening
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CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was used to measure ATP as a indicator of cell viability. The Vero cell line (ATCC CCL81) was grown in Dulbecco’s Modified Eagle Medium (DMEM), High Glucose, GlutaMAX™ (Invitrogen), 10% FBS (Fetal Bovine Serum), and 1x of Penicillin-Streptomycin Solution (100 units/mL of penicillin, 100 µ g/mL of streptomycin). Compounds were solubilized in DMSO (dimethyl sulfoxide) and assayed using a 10-point three-fold serial dilution starting at the highest concentration of 50 µ M. CellTiter-Glo® Reagent (Promega) was added to 96-well plates after 2 days of incubation at 37 °C, 5% CO2. Relative luminescent units (RLU) were measured using Perkin Elmer Wallac 1420 Victor2 plate reader. Inhibition curves were fitted using the Levenberg–Marquardt algorithm. Toxic concentration (TC50) was defined as the concentration of compound that gave 50% inhibition of growth. Selectivity index was calculated as MIC/TC50. For published data (Ananthan et al., 2009 (link)), SI was calculated as IC90/TC50.
4
Mast Cell Calcium Flux Assay
5
Calcium Imaging of Airway Epithelial Cells
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16HBE or HUVEC (1.5 . 104 cells/well) were loaded with 100 μL of the dye loading solution containing Fluo-4 NW dye and probenecid, according to the manufacturer’s instructions (Molecular Probes Fluo-4NWCalcium Assay kit)18 (link). Briefly, the 96-well plate was incubated in the presence or absence of tiotropium at 37 °C for 45–60 min in the dark and ACh (1 mM) was added to the cells. The changes in Fluo-4 NW fluorescence were measured by the Wallac 1420 Victor 2 (Perkin–Elmer, Milan, Italy) at λex 494 nm and λem 516 nm. Calcium mobilization was observed over time (up to 110 s) and analyzed by the Wallac 1420 Software version 3 (Perkin– Elmer Life and Analytical Sciences, Wallac, Milan, Italy). The increase in [Ca2+]i fluorescence was expressed as relative fluorescence units (RFU).
6
Measurement of Intracellular ROS Levels
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The peroxide‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate (DCF‐DA; Molecular Probes, Carlsbad, CA, USA) was used to assess the generation of intracellular ROS as described previously.21 Cells were grown in 6‐well plates and serum‐deprived overnight. Cells were washed with Hanks' balanced salt solution without phenol red and then incubated for 30 min in the dark at 37°C with the same solution containing the peroxide‐sensitive fluorophore DCF‐DA (Molecular Probes) at 5 μmol/L. The DCF‐DA fluorescence was detected at excitation and emission wavelengths of 488 and 520 nm, respectively, as measured with a multiwall fluorescence plate reader (Wallac 1420 Victor2; PerkinElmer Life Sciences, Waltham, MA).
7
ACh Production Measurement in 16-HBE Cells
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ACh production was performed as previously described [30 (link)]. It was measured in protein extracts from cultured 16-HBE cells by a fluorimetric method using a commercial kit (BioVision Research Products, CA, USA, cat. #K615-100). The kit detects choline (Ch) and total choline (TCh) by adding acetylcholine esterase to the reaction that converts ACh into Ch with sensitivity until 50 pmol/well by plotting fluorescence readings (Ex/Em 535/587 nm) against the standard curve. This sensitivity is correspondent to the concentration of 1 μM of TCh or Ch. ACh was evaluated as difference between TCh and Ch. Fluorescence intensity was read using a Wallac 1420 Victor2 multilabel counter (PerkinElmer Life Sciences, Turku, Finland). Results were expressed as pmoli/μg protein.
8
Quantification of Cellular Triglycerides
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Triglycerides were quantified in cell lysates and supernatants using the Triglyceride Assay Kit (Fluorometric, Reducing Samples) (Abcam). Briefly, supernatants were collected, and cells were lysed using the kit provided assay buffer. Fluorescence was measured using a Perkin Elmer Wallac 1420 Victor2 microplate reader. Sample fluorescence was compared to a standard curve to determine the triglyceride concentration in samples.
9
MTT Assay for Cell Viability
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After overnight seeding, 6×103 HeLa cells were attached in 96-well plates and treated with the corresponding eight groups as mentioned earlier for 24 h. The cells were incubated with 0.2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) and maintained for another 4 h. The final product of formazan was dissolved in DMSO, and the optical density value was measured using a Wallac 1420 Victor2 microplate reader (Perkin Elmer, Turku, Finland) at the wavelength of 540 nm. Relative cell viability (%) was calculated by dividing the number of cells treated with each group by the number of cell CTR. Data are shown as mean ± standard deviation (SD) from three separate measurements.
10
Quantitative DNA Fluorescence Assay
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Five to 20 μl of each proteinase K-digested sample was loaded in technical triplicate on an ice-cold 96-well plate. After incubation with heparin (8.3 U ml−1 in PBS; Leo Pharma) and RNase solution (0.05 mg ml−1 in PBS; Sigma-Aldrich), 50 μl of ethidium bromide (25 μg ml−1 in PBS; GIBCO) was added to each sample. Using the Wallac 1420 VICTOR2 (PerkinElmer) apparatus, the ratio between the absorbance at 340 nm (extinction filter) and the absorbance at 590 nm (emission filter), corrected by the background (wells loaded with PBS only), was calculated. Purified calf thymus DNA (Sigma-Aldrich) was used to set the standard curve.
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