Cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and were cultured according to the supplier’s instructions. Briefly, human KB epidermal carcinoma cells were grown in Dulbecco minimal essential medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal calf serum (FCS) and 1% glutamine, 100 UI penicillin, 100 µg/mL streptomycin and 1.5 µg/mL fungizone and maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cell viability was assessed using Promega CellTiter-Blue TM reagent according to the manufacturer’s instructions. Cells were seeded in 96-well plates (5 × 103 cells/well) containing 50 mL growth medium. After 24 h of culture, the cells were supplemented with 50 mL of the studied compound dissolved in DMSO (less than 0.1% in each preparation). After 72 h of incubation, 20 mL of resazurin was added for 2 h before recording fluorescence (λex = 560 nm, λem = 590 nm) using a Victor microtiter plate fluorimeter (Perkin-Elmer, USA). Results are shown in Table 1 .
Victor microtiter plate fluorimeter
Manufactured by PerkinElmer
Sourced in United States
The Victor microtiter plate fluorimeter is a versatile instrument designed for fluorescence-based assays. It can measure fluorescence intensity in microplates, supporting a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter blue reagent, by Promega (3 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Cambrex (1 mentions)
Apo one homogeneous caspase 3 7 assay, by Promega (1 mentions)
5 protocols using victor microtiter plate fluorimeter
1
In Vitro Cytotoxicity Assay for Cancer Cells
2
Cytotoxicity Assay for Cancer Cell Lines
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Human breast
adenocarcinoma
(MDA-MB-231), human nonsmall cell lung carcinoma (A549), and human
cervical carcinoma (HeLa) cells were grown in DMEM medium supplemented
with 115 units/mL of penicillin G, 115 μg/mL of streptomycin,
and 10% fetal bovine serum (all from Life Technologies, Grand Island,
NY). Cells were seeded in 96-well plates (5 × 103 cells/well)
containing 50 μL of growth medium for 24 h. After medium removal,
100 μL of fresh medium containing individual analogue compounds
at different concentrations was added to each well and incubated at
37 °C for 72 h. After 24 h of culture, the cells were supplemented
with 50 μL of analogue compounds dissolved in DMSO (less than
0.25% in each preparation). After 72 h of incubation, 20 μL
of resazurin was added for 2 h before recording fluorescence at 560
nm (excitation) and 590 nm (emission) using a Victor microtiter plate
fluorimeter (PerkinElmer, USA). The IC50 was defined as
the compound concentration required to inhibit cell proliferation
by 50% in comparison with cells treated with the maximum amount of
DMSO (0.25%) and considered as 100% viability.
adenocarcinoma
(MDA-MB-231), human nonsmall cell lung carcinoma (A549), and human
cervical carcinoma (HeLa) cells were grown in DMEM medium supplemented
with 115 units/mL of penicillin G, 115 μg/mL of streptomycin,
and 10% fetal bovine serum (all from Life Technologies, Grand Island,
NY). Cells were seeded in 96-well plates (5 × 103 cells/well)
containing 50 μL of growth medium for 24 h. After medium removal,
100 μL of fresh medium containing individual analogue compounds
at different concentrations was added to each well and incubated at
37 °C for 72 h. After 24 h of culture, the cells were supplemented
with 50 μL of analogue compounds dissolved in DMSO (less than
0.25% in each preparation). After 72 h of incubation, 20 μL
of resazurin was added for 2 h before recording fluorescence at 560
nm (excitation) and 590 nm (emission) using a Victor microtiter plate
fluorimeter (PerkinElmer, USA). The IC50 was defined as
the compound concentration required to inhibit cell proliferation
by 50% in comparison with cells treated with the maximum amount of
DMSO (0.25%) and considered as 100% viability.
3
Endothelial Cell Viability Assay
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Cancer cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics (Lonza; Walkersville, MD, USA) and were cultured as described in the Supplementary Information and according to the supplier's instructions. For the proteomics and transcriptomics analysis 6.2×106 sensitive and insensitive cells were plated in petri dishes and cultured as described in the Supplementary Information. After 24 h medium is removed and changed with fresh medium containing or not 5μM fumagillin. Cells were cultured for additional 24h before collecting them for further analysis. Cell viability was assessed with the Promega CellTiter-Blue™ reagent, according to the manufacturer's instructions. After 24 h of culture, the cell medium was supplemented with 50 μL of the test compound dissolved in DMSO (<0.1% in each preparation). After incubation for 72 h, we added 20 μL resazurin and incubated the mixture for a further 2 h before recording fluorescence (λex=560 nm, λem=590 nm) in a Victor microtiter plate fluorimeter (Perkin Elmer, USA). Experiments were performed in triplicate.
4
Cytotoxicity Assay for HUVECs
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Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (Lonza; Walkersville, MD, USA) and cultured according to the supplier’s instructions. Briefly, HUVECs from three to six passages were subcultured to confluence onto 0.2% gelatin-coated tissue culture flasks in endothelial cell growth medium (EGM2) containing growth factors and 2% FCS. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cell viability was assessed using Promega (Promega France, Charbonnière-les-Bains, France) CellTiter-BlueTM reagent according to the manufacturer’s instructions. Cells were seeded in 96-well plates (5 × 103 cells per well) containing 50 mL growth medium. After 24 h of culture, the cells were supplemented with 50 mL of test compound dissolved in DMSO (<0.1% in each preparation). After incubation for 72 h, 20 mL resazurin was added for 2 h before recording fluorescence (λex = 560 nm, λem = 590 nm) using a Victor microtiter plate fluorimeter (PerkinElmer France, Villebon-sur-Yvette, France). IC50 values correspond to the concentration of test compound that elicited a 50% decrease in fluorescence for drug-treated cells relative to untreated cells. Experiments were performed in triplicate. Fumagillin was used as a positive control and showed an IC50 of 0.006 ± 0.002 μM.
5
Caspase-3/7 Apoptosis Assay in U87 Cells
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Apoptosis was measured by the Apo-one homogeneous caspase-3/7 assay (Promega Co, WI) according to the manufacturer's recommendations. Briefly, U87 cells were subcultured on a 96-well plate with 5 × 10 4 cells/well in 100 μL medium. After 24 h of incubation, the medium in the 96-well plate was discarded and replaced with medium containing 4b-d at a concentration of 25 nM or 0.1% DMSO (as negative control). The U87 cells were incubated for 24 h, each well then received 100 μL of a mixture of caspase substrate and Apo-one caspase 3/7 buffer. After 1 h of incubation, the fluorescence of sample was measured using a Victor microtiter plate fluorimeter (Perkin-Elmer, USA) at 527 nm.
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