Cells (5×104/well) were cultured in 96 well plates. After 24 h incubation in DMEM without serum, treatments were added to each plate, as indicated. After 24 h of treatment, 50 µl of XTT reagent was added to each plate according to the manufacturer’s protocol (Biological Industries) for 2–5 h following solubilization, the orange color was detected by spectrophotometer at 450 nm.
Xtt reagent
Manufactured by Sartorius
Sourced in Israel
The XTT reagent is a colorimetric assay used to measure cell viability and proliferation. It functions by detecting metabolically active cells, which convert the yellow tetrazolium salt XTT into an orange formazan product.
Automatically generated - may contain errors
Lab products found in correlation
Synergy 2 multi detection microplate reader, by Agilent Technologies (1 mentions)
Hypoxia incubator chamber, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Elisa reader, by Agilent Technologies (1 mentions)
Multiskan ex, by Thermo Fisher Scientific (1 mentions)
9 protocols using xtt reagent
1
XTT Viability Assay Protocol
2
Evaluation of Inhibitors on Mouse MSCs
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Mouse mesenchymal stem cells (MSCs) were obtained by flushing the bone marrow from femurs and tibias of 10-weeks-old mice as described in the cell binding assay section. Cells were cultured in MesenCult MSC Basal Medium with 1/5 MSC stimulatory supplements, 50 U/L penicillin-streptomycin, and 10 mg/mL gentamycin and kept in a Hypoxia Incubator Chamber (Thermo Fisher Scientific, Waltham, MA, USA) under 5% oxygen. Then, 7,000 cells were plated in each well of a 96-well plate and held for 48 h in an incubator at 37 °C in the presence of different concentrations of inhibitors (50 nM, 1 μM, and 5 μM). After 48 h, the XTT reagent (Biological Industries, Israel) was added to the cells according to manufacturer's protocol for 2 h at 37 °C. The plate was rocked gently, and the absorbance was measured at 450 nm and 650 nm for background subtraction with a Synergy 2 multidetection microplate reader (Biotek, Winooski, VT, USA). Each condition was repeated three times, and the blank absorbance measurements and the background absorbance were subtracted from the sample's absorbance.
3
Cell Viability Assay with Botanical Extracts
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The cells were plated at a density of 3,000 cells/well in triplicate over 96-well plates in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 µg/ml penicillin/streptomycin (Biological Industries, Beit Ha-Emek, Israel) and allowed to attach and grow overnight at 37°C, in a 5% CO2 atmosphere. The medium was replaced with a fresh treatment-containing medium, and the cells were propagated for an additional 48 h in the same conditions. In experiments with pyruvate and N-acetyl-cysteine (NAC), 1 mM pyruvate or 10 mM NAC were added to the medium together with botanical extracts, mixed and then added to the cells. An XTT viability test was performed by replacing the medium with fresh RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 µg/ml penicillin/streptomycin, to prevent interference of treatment color with XTT reagent signal, and adding XTT reagent (Biological Industries) for incubation for 2–3 h. The blank measurement was subtracted from each reading, and all values of treated cells were divided by control (PBS-treated cells) values in each experiment (control=1). The resulting signal was measured by an enzyme-linked immunosorbent assay reader. Each experiment was repeated at least three times.
4
Anoikis Assay for Cell Viability
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The anoikis activity of the cells was investigated using a CytoSelect 96-well anoikis assay kit (Cell Biolab, San Diego, USA). Briefly, 1 × 104 cells were seeded onto the nonadherent poly-HEMA-coating well under regular culture conditions, and cell viability was detected 24 h later using XTT reagent (Biological Industries). The control well contained 1 × 104 cells without poly-HEMA coating. The viability ratio of cells grown in the two different wells was calculated using ODanoikis well/ODcontrol well. Each experiment was triplicated.
5
Cytotoxicity of C9-PEX on MCF7-MMP9 Cells
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MCF7-MMP9 cells (1.2 × 104) were grown overnight at 37°C under 5% CO2 on a 96-well plate. Thereafter, the cells were treated for 24 h with different concentrations of C9-PEX, namely, 10 µM, 5 µM, 2 µM, 1 µM, 100 nM or 10 nM, in serum-free medium. XTT reagent (Biological Industries, Israel) was added according to the manufacturer's instructions, and the absorbance was measured after 2 h at wavelengths of 450 nm and 690 nm.
6
XTT Cell Proliferation Assay
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Cell proliferation assay was performed using 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) reagent (Biological Industries, Kibbutz Beit-Haemek, Israel). Briefly, 1 × 103 cells were seeded in a 96-well plate, and the medium was replaced every day for one week experiments. The OD ratio was calculated from the specific absorbance at 450 nm adjusted with a nonspecific absorbance at 650 nm in an ELISA reader (BioTek, Winooski, VT, USA). The blank well contained medium only. Each experiment was triplicated.
7
Viability Assay of DNA-Damaged Cells
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MCF10A or HMECs were plated at 60–70% confluence in a 96-well culture dish (5 × 103 cells in 0.1 mL complete growth medium per well), incubated over night for attachment, and treated with the indicated DNA damaging agents for 24−48 h. For cell viability assay, 0.05 mL of XTT reagent (Cell proliferation kit, Biological Industries, Beit Haemek, Israel) were added to each well. The colorimetric reaction was developed within 2–4 h incubation at 37 °C and absorbance was measured at a wavelength of 450–500 nm using a plate reader (Multiskan Ex, Thermo Fisher Scientific Inc., Waltham, MA, USA).
8
HepG2 Cell Viability Assay with Rotenone and Antibody Treatment
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Human hepatocellular carcinoma cells (HepG2) were seeded, 2.5 × 104 cells/well in 96-well plate, containing Dulbecco’s modified Eagle’s medium with 1 g/L D-glucose (low glucose), at 37°C, 5% CO2 for 6 h and later treated with 5 µM Rotenone at 37°C, 5% CO2 for 4 h. Then, the medium containing Rotenone was replaced by fresh medium, and the plate was kept at 37°C, 5% CO2 overnight. Next, the cells were treated with or without different antibodies (as indicated) at a final concentration of 1 µM for 22 h. After incubation, 50 µl/well XTT reagent (Biological industries, Israel) was added according to suppliers’ recommendations at 37°C, 5% CO2 for 2 h. Plate was analyzed with an EMax Plus Microplate Reader at 450 and 630 nm. At all stages, volume was 100 µl/well unless mentioned otherwise.
9
Cytotoxicity Evaluation of Microspheres
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Fibroblast NIH-3T3 and L929 cells were used for cytotoxicity assays. Media and serum were purchased from Gibco®, Thermo Fisher Scientific Inc., Waltham, MA, USA. NIH-3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% bovine serum. L929 cells were cultured in minimal essential medium alpha medium (MEMα) containing 10% horse serum.
NIH-3T3 and L9292 cells cultured in pure medium were set as control groups. The positive control was 15 vol.% dimethyl sulfoxide (DMSODMSO), and the negative control was high-density polyethylene (HDPE) extract to verify sample sterility. The microspheres were soaked in the medium at a ratio of 1:10 for 24 h as sample extracts. The cells were inoculated in a 96-well culture plate at a concentration of 1 × 104 and cultured for 24 h. XTT reagent (Biological Industries, Kibbutz Beit Haemek, Israel; 50 μL/wells) was used to measure cell viability at OD492 by ELISA reader. Cell morphology was observed under an optical microscope (IVM-3AFL, SAGE VISION Co., Ltd., New Taipei City, Taiwan).
NIH-3T3 and L9292 cells cultured in pure medium were set as control groups. The positive control was 15 vol.% dimethyl sulfoxide (DMSODMSO), and the negative control was high-density polyethylene (HDPE) extract to verify sample sterility. The microspheres were soaked in the medium at a ratio of 1:10 for 24 h as sample extracts. The cells were inoculated in a 96-well culture plate at a concentration of 1 × 104 and cultured for 24 h. XTT reagent (Biological Industries, Kibbutz Beit Haemek, Israel; 50 μL/wells) was used to measure cell viability at OD492 by ELISA reader. Cell morphology was observed under an optical microscope (IVM-3AFL, SAGE VISION Co., Ltd., New Taipei City, Taiwan).
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