The assay, based on the staining of living cells by neutral red, was performed according to the protocol described by Borenfreund and Puerner [19 (link)]. After incubation, the medium containing the compound was removed and the cells were washed with PBS. Then, a 100 µL/well of NR solution (50 µg/mL) was added for 3 h. After that time, the cells were washed with PBS. The dye from viable cells was released by extraction with a mixture of acetic acid, ethanol, and water. After 10 min of shaking, the absorbance of the dissolved NR was measured at 540 nm in a microplate reader (LabSystems Multiskan RC, Thermo Fisher Scientific, Waltham, MA, USA) using a blank as a reference. Cytotoxicity was expressed as a percentage of the negative control (0.1% DMSO).
Labsystems multiskan rc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LabSystems Multiskan RC is a microplate reader designed for absorbance-based assays. It measures the optical density of samples in microplates, enabling quantitative analysis of a variety of assays such as enzyme-linked immunosorbent assays (ELISA), cell-based assays, and colorimetric biochemical assays.
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Lab products found in correlation
5 protocols using labsystems multiskan rc
1
Neutral Red Cytotoxicity Assay
2
Membrane Integrity Assessment by LDH Release
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The integrity of the plasma membrane was assessed using the test of lactate dehydrogenase (LDH) release [22 (link)], which was monitored using the commercially available Cytotoxicity Detection Kit (LDH) (Roche Diagnostics, Warsaw, Poland). The medium (100 µL/well) without cells was transferred into the corresponding wells of an optically clear 96-well flat bottom microplate and a 100 µl reaction mixture was added to each well. Then, the plates were incubated for 30 min at room temperature in darkness. After that time, a 50 µL/well 1 M HCl was added to stop the reaction. The absorbance was measured at 492 nm in a microplate reader (LabSystems Multiskan RC, Thermo Fisher Scientific, Waltham, MA, USA) using a blank as a reference.
3
Coomassie Brilliant Blue Cell Proliferation Assay
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The assay was based upon staining total cellular protein (proliferation) [20 (link)]. After incubation, the medium containing the compound was removed and 100 µL of Coomassie Brilliant Blue R-250 dye were added to each well. The plate was shaken for 10 min. Then, the stain was removed and the cells were rinsed twice with 100 µL of washing solution (glacial acetic acid/ethanol/water). After that, a 100 µL portion of the desorbing solution (1 M potassium acetate) was added and plates were shaken again for 10 min. The absorbance was measured at 595 nm in a microplate reader (LabSystems Multiskan RC, Thermo Fisher Scientific, Waltham, MA, USA) using a blank as a reference. Cytotoxicity was expressed as a percentage of the negative control (0.1% DMSO).
4
Evaluating Cell Metabolic Activity
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The metabolic activity of living cells was assessed by measuring the activity of dehydrogenases [21 (link)]. After incubation of the cells with the compounds, a 10 μL portion of the MTT solution (5 mg/ml in PBS) was added to each well of the 96-well plates and incubated. After 3 h, the MTT solution was removed and the intracellular formazan crystals were dissolved in 100 µl DMSO. The plate was shaken for 15 min at room temperature and transferred to a microplate reader (LabSystems Multiskan RC, Thermo Fisher Scientific, Waltham, MA, USA) to measure the absorbance at 570 nm, using blanks as a reference. Cytotoxicity was expressed as a percentage of the negative control (0.1% DMSO).
5
Cell Viability with Silica Nanoparticles
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Cell viability and proliferation of CCD-25SK cells, following incubation with the Si supplemented media, was quantitatively evaluated with the fluorescent and non-toxic cell dye Resazurin (Invitrogen, Life Technologies UK). Briefly, the Si supplemented media and controls were removed after 24 and 72 h incubation and the cells rinsed with phenol red-free MEM. Cells were then incubated with phenol red-free MEM containing 10% (44 μM) Resazurin for 6 h at 37˚C in a humidified atmosphere containing 5% CO2. Aliquots of the media were then transferred to a flat-bottom 96-well plate and the absorbance measured at 540 nm and 595 nm on an optical plate-reader (Labsystems Multiskan RC, Thermo Scientific Inc, USA). To check for false positive results originating from the silica NPs, a control containing just particles was incubated with the Resazurin dye, and this gave negligible absorbance values.
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