Cells were seeded in 384 well plates at a density of 5000 cells per well in 25 μL medium. Drug treatment was performed 2 hours post-thawing by adding increasing doses of drug from a 3-fold dilution series to test 11 different concentrations. Each dose was added in four replicate wells, and the plate was incubated at 37°C. Following selinexor treatment, cell viability was assessed 48 h post-drug treatment by adding PrestoBlue reagent (ThermoFisher) and measuring fluorescence using a SpectraMax i3 multi-mode plate reader (Molecular Devices). Following nutlin-3a treatment, cell viability was assessed 72 h post treatment by adding Cell Titer Glo 2.0 (Promega) and measuring luminescence in a SpectraMax i3 multi-mode plate reader. For drug combinations of selinexor and MK-2206, or selinexor and nutlin-3a, cells were seeded in 384-well plates followed by drug treatment using a 3-fold serial dilution of selinexor and a 5-fold serial dilution of either MK-2206 or nutlin-3a. Cell viability was assessed 72 h post drug treatment by adding Cell Titer Glo 2.0 (Promega) and measuring luminescence in a SpectraMax i3 multi-mode plate reader.
Spectramax i3 multi mode plate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax i3 multi-mode plate reader is a versatile laboratory instrument designed for a wide range of absorbance, fluorescence, and luminescence assays. It features a monochromator-based optical system, allowing for flexible wavelength selection and optimization of detection parameters.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo 2, by Promega (1 mentions)
Prestoblue reagent, by Thermo Fisher Scientific (1 mentions)
Enliten atp bioluminescence detection kit, by Promega (1 mentions)
Texas red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
G1393, by Merck Group (1 mentions)
C7661, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
7 protocols using spectramax i3 multi mode plate reader
1
Cytotoxicity Assay for Combination Treatments
2
Cytotoxicity of GSLL on Nasopharyngeal Carcinoma
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Human nasopharyngeal carcinoma CNE1 and CNE2 cells were purchased from the Sun Yat-sen University of Medicinal Sciences (China). The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum and 100 U/mL penicillin and streptomycin, and maintained at 37 °C in a humidified incubator under 5 % CO2. The cells were seeded onto Corning® Costar® 96-Well plates (Code: CLS3595; Sigma-Aldrich) overnight. Different concentrations of GSLL were added to the cells and incubated at 37 °C for 24, 48, or 72 h. The effects of addition of 62.5 mM glucose or mannose on the GSLL treatment of CNE1 cells were examined to investigate the inhibitory effects of the carbohydrates on the action of GSLL on CNE1 cells. Briefly, after the wells were washed with PBS, 25 µL of MTT (5 mg/mL) in PBS was added and incubated for 4 h. Next, 150 µL of DMSO was added to the wells, and the optical density at 580 nm was recorded with a SpectraMax i3 Multi-mode Plate Reader (Molecular Devices, USA). The IC50 values for GSLL treatment of the cells were determined as the GSLL concentrations causing 50 % inhibition of the cells.
3
Analytical Reagents and Cell Culture Protocol
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This investigation used chemicals and reagents of analytical grade obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA) and Merck KGaA (Darmstadt, Germany). All other reagents otherwise not stated were of chemical purity. Materials used in cell culture procedures were acquired from Kasvi (São José dos Pinhais, PR, Brazil), Corning Inc. (Corning, NY, USA), and Vitrocell Embriolife (Campinas, SP, Brazil). Western blot and molecular biology reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA), Merck KGaA, and Sigma-Aldrich Inc. All measurement analyses were carried out using a SpectraMax® i3 Multimode Plate Reader (Molecular Devices, Sunnyvale, CA, USA). The fluorescent images were captured using an Olympus Fluorescent Microscope (Fluoview FV101, Olympus, Tokyo, Japan).
4
ATP Bioluminescence Assay in U2OS Cells
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Eight thousand U2OS wild-type cells per well were seeded in a 96-well plate. The next day, cells were treated for 24 hours. Then the supernatant was collected, centrifuged and transferred to a white bottom 96-well plate. Enzyme and substrate from ENLITEN ATP Bioluminescence Detection Kit (FF2000; Promega, Madison, Michigan, USA) were added. ATP-dependent substrate conversion was measured by luminescence at 560 nm with a Spectramax I3 multimode plate reader (Molecular Devices).
5
Dextran Extravasation Quantification Protocol
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Dextran extravasation was performed as described previously35 (link),36 (link) with modification. Mice were injected intraperitoneally with 100 μL of fluorescently conjugated 10-kDa (Alexa Fluor-555; Invitrogen) and 70-kDa (Texas Red; Invitrogen) dextran tracers at a concentration of 3.75 mg/mL and returned to their home cages for 30 minutes to allow the dextrans to circulate. Animals were then transcardially perfused with TBS. Cortices were dissected, weighed, and incubated in formamide for 48 hours at 56 °C. Dye fluorescence was then measured using a SpectraMax i3× Multi-Mode Plate Reader (Molecular Devices) at the appropriate emission and excitation wavelength and normalized to tissue weight.
6
Cytotoxicity Assay of EOPc on Melanoma
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The cytotoxicity assay of EOPc on SK-MEL-28 and CCD-059sk was made with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma Aldrich, MO, USA) according to Mosmann (1983) (link). Briefly, both cells were separately seeded in 96-well plates, in 4 replicates, at densities of 1 × 105 cells/well. The cells were treated with different concentrations of EOPc. After the exposure times, the supernatant was removed, and the cells were washed once with phosphate-buffered saline (PBS) (0.1 mol/L, pH 7.4) to avoid any interference from the compound used in the treatment. The MTT reagent (5 mg mL−1) dissolved in PBS was added, and plates were incubated for 2 h at 37 °C. Subsequently, the supernatant was discarded, and 200 µL of DMSO was added to dissolve the formazan crystals generated by reduction of tetrazolium salts by dehydrogenases and reductases. The absorbance was measured at 570 nm using a SpectraMax® i3 Multimode Plate Reader (Molecular Devices, San Jose, CA, USA) and the results were expressed as percentage (%) of cell viability relative to control.
7
Cell Adhesion Assay Protocol
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Cell culture plates (48-well) were coated with 0.1% gelatin (G1393, Sigma Aldrich) for 30 min at 37 °C or 10 µg/mL collagen type I from rat tail (C7661, Sigma Aldrich) overnight at 4 °C. Gelatin was diluted in distilled and sterilized water and collagen I was diluted in phosphate-buffered saline (PBS). Prior to the experiment, plates were incubated with 0.25% BSA (Sigma Aldrich) in PBS at 37 °C for 30–90 min to block non-specific binding. In parallel, the C2C12α10, U3054MG, and U3046MG cells were harvested, suspended into a single cell suspension in Hank’s Balanced Salt Solution (HBSS). The cells were pre-incubated for 30 min in the presence or in the absence of antibodies at a concentration of 10 µg/mL at 4 °C. The cells were then allowed to attach to the pre-coated plates for 40–60 min at 37 °C, and unattached cells were removed by washing with HBSS. Adherent cells were fixed with ethanol, stained with 0.09% crystal violet (Sigma Aldrich), and the absorbed dye was extracted by 10% acetic acid (Sigma Aldrich). The extraction was analyzed by measuring the optical density (OD) at 590 nm in the SpectraMax i3 multi-mode plate reader (Molecular Devices, San Jose, CA, USA). All the results were expressed as the percentage of adherent cells considering the untreated as 100%.
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