Cell viability was determined by the XTT assay. Briefly, 20,000 C4-1 wildtype and mutant cells per well were seeded in flat-bottomed 96-well plates in DMEM medium with 10% fetal bovine serum (FBS) and incubated overnight at 37 °C, 5% CO2. Then, a series of serial dilutions (1:2) of CIGB-300 (31.25–500 μM) and CX-4945 (3.125–50 µM) were added in triplicate. After 48 h, 50 µL of XTT labeling mixture (5 mL XTT labelling reagent + 0.1 mL electron coupling reagent) (Roche, Basel, Switzerland) was added to each well, and the cells were further incubated for 4 h at 37 °C. Following the incubation period, absorbance at 490 nm was read using an ELISA plate reader (BioRad, Watford, UK). The percentage of cell viability was calculated using the following formula: Cell viability (%) = ((OD of treated cells)/(OD of cell control)) × 100. The half-cytotoxic concentration (CC50) was estimated from the fitted dose–response curves using the CalcuSyn software (Biosoft, Cambridge, UK).
Xtt labeling mixture
Manufactured by Roche
Sourced in Germany
The XTT labeling mixture is a laboratory reagent used for the colorimetric detection and quantification of cell viability and proliferation. It is a tetrazolium compound that is reduced by metabolically active cells, producing a colored formazan product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
96 well microtiter plate, by Thermo Fisher Scientific (2 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Calcusyn software, by Biosoft (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Prism 6, by GraphPad (1 mentions)
Recombinant human il 6, by R&D Systems (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
6 protocols using xtt labeling mixture
1
Cell Viability Determination by XTT Assay
2
Neuronal Culture Hypoxia Protocol
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Primary neuronal cultures derived from embryonic day 14–18 mouse cortices were grown to 80% confluency in neural basal media supplemented with B27 (2%) and penicillin/streptomycin (1%), as previously described (Kintner et al., 2010 (link)). Astrocytic and microglial contamination was excluded based on the absence of GFAP+ and CD11b+ cells when stained by immunocytochemistry. For OGD, media was replaced with neural basal media with or without glucose and placed in a hypoxic chamber or under normoxic conditions for 2 h at 37°C. Afterward, cells were lysed and mRNA isolated using RNeasy mini kit (QIAGEN). For XTT viability assay, Neuro2A underwent OGD and were treated with dose curve of IL-21 immediately after return to normoxic media and incubated for 4 h at 37°C. XTT labeling mixture (50 µl per well; Roche) was added according to manufacturer’s instructions, and at18 h fluorescence was read on a GENious Microplate reader (Tecan). Cell number was calculated using a standard curve of known untreated cells kept under normoxic conditions.
3
Cytotoxicity Assay with Drugs and Inhibitors
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Cells were seeded (4×103 cells/well) in 96-well microtiter plates (Nunc, Wiesbaden-Biebrich, Germany) in 100 µl IMDM with 10% FBS in quintuplicate. After 24 h, culture medium was changed to IMDM with 10% FBS and different concentrations of drugs or inhibitors. For drug IC50 detection, cells were treated with different dosages of cisplatin, 5-FU or docetaxel directly. Cells were also cultured in the presence of human recombinant IL-6 or human IL-6R/gp130 neutralizing antibody (all from R&D Systems, Minneapolis, MN, USA) for 24 h, followed by 5 µM cisplatin treatment to examine changes in drug resistance. Cells were further grown for 72 h, before incubated in 50 µl XTT labeling mixture (Roche Molecular Biochemicals, Mannheim, Germany) for 4 h, and then scanned at 450 nm. IC50 was calculated using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).
4
Cytotoxicity Assay for MDA-MB-231 Cells
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MDA-MB-231 cells were seeded in 96-well plates at a concentration of 5 × 103 cells per well and incubated at 37 °C for 6 h. Cells were then exposed to a series of concentrations of single drug-loaded cMLVs for 48 h. Fifty microliters of XTT labeling mixture (Roche Applied Science) was then added. After incubation at 37 °C for 4 h, the absorbance of the solution was measured at 570 nm using a microplate reader (Molecular Devices) to determine the optical density value. Cell viability was calculated by subtracting absorbance values obtained from media-only wells from drug-treated wells and then normalizing to the control cells without drugs. Data are given as mean ± SD of three independent measurements.
5
Cytotoxicity Assay with Cisplatin
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Cells were seeded at a density of 4000 cells per well in 96-well microtiter plates (Nunc, Wiesbaden-Biebrich, Germany) in 100 μl culture medium with 10 % FBS per well in quintuplicate. After 24 h, culture medium was exchanged to medium with 10 % FBS and different concentration of cisplatin or growth factors. Cells were further grown for 72 h, before incubated in 50 μl XTT labeling mixture (Roche Molecular Biochemicals, Mannheim, Germany) for four h, and then scanned at 450 nm in an Epoch Microplate Spectrophotometer (BioTek, Winooski, USA).
6
Biofilm Antimicrobial Susceptibility Testing
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The MBECs of bacterial biofilm cultures for amikacin and ceftazidime were determined according to the method of Amorena et al using the XTT (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) colorimetric assay with some modifications [19 (link)]. Briefly, biofilms were established in the wells of a flat-bottomed polystyrene 96-well microtiter plate (Tissue culture plate 96 wells, JET BIOFIL, Canada). After incubation of bacterial biofilms with 100 μL of serial dilutions of antibiotics at 37°C for 20 h, 50 μL of fresh XTT labeling mixture (Roche, Germany) was added to each well and subsequently incubated for 1 h at 37°C in the dark conditions [20 (link)]. The lowest concentration of the antibiotic that inhibited re-growth of the bacteria from the treated biofilm was defined as the MBEC value [21 (link)]. This test was conducted 3 times. This experiment was performed on 3 strains 1, 2 and 4, because they were susceptible to ceftazidime and amikacin in planktonic state, but strain 3 that was resistant to amikacin and strain 5 that was resistant to both amikacin and ceftazidime, were not involved in the experiment.
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