CCRF-CEM and MDA MB231 cells were treated with FK866, CHS-828 (200484-11-3, Cayman Chemical), oligomycin A (75351, Sigma), 2-deoxyglucose (D8375, Sigma), GSK2837808A (GSK) (5189, Tocris), JPH203 (a selective L-type amino acid transporter), and l -asparaginase (11185, Sigma) for 48 h. JPH203 was kindly obtained from Dr. Peyron [30 (link)]. In vitro drug sensitivity was assessed by the colorimetric methyl-thiazolyltetrazolium (MTT) assay (sigma), XTT proliferation kit (sigma), and OZBlue Cell Viability kit (OZbiosciences). Combination treatment of FK866 with JPH203 was performed and 48 h after combination treatment, Cell viability was determined using XTT assay (Invitrogen) and DAPI staining. Percentage of cell death was subjected for drug combination analysis as described by combination index (CI). CI was analyzed using CompuSyn software V1.0 by the method of Chou and Talalay [31 (link)]. CI < 1 indicates drug synergistic effect, CI > 1 indicates drug antagonistic effect, and CI = 1 indicates drug additive effect.
Xtt assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The XTT assay is a colorimetric method used to measure cell viability and proliferation. It involves the reduction of the tetrazolium salt XTT to an orange-colored formazan product, which can be quantified spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
L asparaginase, by Merck Group (1 mentions)
Jph203, by Merck Group (1 mentions)
Chs 828, by Cayman Chemical (1 mentions)
Jph203, by Bio-Techne (1 mentions)
2 deoxyglucose, by Merck Group (1 mentions)
Oligomycin a, by Merck Group (1 mentions)
Ozblue cell viability kit, by Ozyme (1 mentions)
Infinite f50, by Tecan (1 mentions)
Idelalisib, by Selleck Chemicals (1 mentions)
Byl719, by Selleck Chemicals (1 mentions)
Imark microplate absorbance reader, by Bio-Rad (1 mentions)
96 well plate, by Greiner (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Ultraculture medium, by Lonza (1 mentions)
Moflo astrios flow cytometer, by Beckman Coulter (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Celltiter glo kit, by Promega (1 mentions)
7 protocols using xtt assay
1
Evaluation of Anticancer Drug Synergy
2
Microglia-Conditioned Medium Effects on Neuronal Viability
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The effect of conditioned medium obtained from microglia on the viability of HT22 cells was measured using the XTT assay (Invitrogen). BV2 cells were pre-treated with tiliroside (2–6 μM) for 30 min and stimulated with LPS (100 ng/ml)/IFNγ (5 ng/ml) for 24 h. Stimulation was terminated by collecting conditioned medium from the cells and centrifuged and stored at − 80 °C. HT22 hippocampal neurons were seeded at a density of 2 × 105 cells/ml in 96-well cell culture plates and incubated at 37 °C. When cells reach confluence, the culture medium was removed and replaced with 100 μl of conditioned medium and further incubated for 24 h. Stimulation was terminated by adding 25 μl of XTT/PMS solution and incubated for 2 h at 37 °C, followed by gentle shaking for few seconds to distribute the orange colour before absorbance was read at 450 nm with a plate reader (Infinite F50, Tecan).
3
Cytotoxicity of Idelalisib and BYL719 in Cell Lines
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The cell lines MKL-1, MKL-2, WaGa, PeTa, MCC13, MCC26 and REH were treated with idelalisib (Selleckchem, Germany) and BYL719 (Selleckchem, Germany). Both agents were dissolved in DMSO. Following concentrations were used for the treatments: 10 nM, 100 nM, 500 nM, 1 µM, 12.5 µM, 25 µM, 50 µM and 100 µM.
The cells were incubated in a 96 well plate (Greiner Bio-One, Austria) for 120 h, in Gibco® RPMI 1640 medium (Life-science) with 10% fetal calf serum (FCS) (Gibco®, Thermo Fisher Scientific) in an incubator at 37° C and 5% CO2. The effect of the drug on the cell viability was assessed by the XTT assay (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. The read out of the XTT assay was done with the iMark™ Microplate Absorbance Reader (BIO-RAD).
To determine the effect of idelalisib on the PI3K pathway the phosphorylation of AKT was assessed by Western blotting. Therefore, the cells were treated with 0.2% DMSO, 1 µM, 12.5 µM and 25 µM of idelalisib for 72 h.
The cells were incubated in a 96 well plate (Greiner Bio-One, Austria) for 120 h, in Gibco® RPMI 1640 medium (Life-science) with 10% fetal calf serum (FCS) (Gibco®, Thermo Fisher Scientific) in an incubator at 37° C and 5% CO2. The effect of the drug on the cell viability was assessed by the XTT assay (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. The read out of the XTT assay was done with the iMark™ Microplate Absorbance Reader (BIO-RAD).
To determine the effect of idelalisib on the PI3K pathway the phosphorylation of AKT was assessed by Western blotting. Therefore, the cells were treated with 0.2% DMSO, 1 µM, 12.5 µM and 25 µM of idelalisib for 72 h.
4
Evaluating Metabolic Activity Through Cryopreservation
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Cell metabolic activity was evaluated with and without the cryopreservation of the constructs by XTT assay (Thermo Fisher Scientific Inc., Waltham, MA, USA). The assay is based on the cleavage of the tetrazolium salt XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) in the presence of an electron-coupling reagent, producing a soluble formazan salt. The concentration of the formazan product is directly proportional to the number of metabolically active cells. Briefly, constructs were washed three times with PBS and then transferred to a new cell culture plate to discard the cells that could be attached to the bottom of the wells. Afterwards, the constructs were incubated at 37 °C for 2 h with 500 µL of the XTT labelling mixture (0.4 mg/mL) in complete cell culture medium. Then, 150 µL of the supernatant per sample was transferred to a 96-well cell culture plate and the optical absorbance (OA) was quantified by spectrophotometry at 450 nm with a plate reader. Results are reported as relative fold change in comparison with OA measured after 3 days of cell culture without cryopreservation.
5
Astrocyte and Neuronal Viability Assay
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Astrocyte viability was determined following pretreatment with drugs or vehicle using the XTT assay (Thermo Scientific). Following a 2‐h incubation with XTT, absorbance was read at 450 nm using a microplate reader (Cytation5®, BioTek). Neuronal viability was assayed following 48‐h incubation with resting or activated astrocyte‐conditioned media. For generation of astrocyte‐conditioned media, astrocytes were grown and treated in serum‐free ultraculture medium (Lonza). Proliferation was assessed by CFSE staining of astrocyte cultures. Briefly, astrocytes were serum starved for 24 h to induce synchronization of cell cycle. Astrocytes were then labeled with 5 μmol/L CFSE (Molecular Probes) for 15 min prior to being returned to serum‐containing medium for 72 h in the presence of treatment conditions. Cells were harvested and fluorescence intensity was measured by flow cytometry using the MoFlo® Astrios™ flow cytometer (Beckman Coulter, Inc.). Proliferation was quantified by loss of CFSE fluorescence.
6
Propagation and Titration of Enteroviruses
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HeLa and RD cells were maintained in DMEM high glucose modification supplemented with pyruvate and 10% FBS. Retrovirus packaging cell line GP2-293 was maintained in DMEM high glucose modification supplemented with 10% FBS. Cell viability was determined using either CellTiter-Glo kit (Promega) or XTT assay (Thermo Fisher) that detect the level of cellular ATP, or the activity of the mitochondrial respiratory chain enzymes, respectively, according to the manufacturers’ recommendations. Poliovirus type I (Mahoney) and Coxsackie virus B3 (Nancy) were rescued using the plasmids pXpA and p53CB3/T7 coding for the full-length viral cDNAs under the control of a T7 promotor kindly provided by Dr. Raul Andino (University of California, San Francisco) and Prof. van Kuppeveld (University of Utrecht, the Netherlands), respectively. The viruses were propagated in HeLa cells, viral titers were determined by plaque or TCID50 assays on RD cells grown on 6- or 96-well plates, respectively, using 10x dilutions of the virus preparations. TCID50 titers were calculated using Kärber’s formula [119 ].
7
Cell Proliferation Assay in DMEM
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Cells were seeded in complete medium on 96-well plates (1 × 104 cells/cm2). After 24 h, the medium was replaced with serum-free low-glucose DMEM (negative control), low-glucose DMEM with 10% serum (positive control), and low-glucose DMEM with 10% CM. After 5 days, the cell proliferation was evaluated by an XTT assay (Thermo Fisher Scientific, Waltham, MA, USA). The data are given as percentage of the positive control.
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