Murine melanoma cell lines 4C11− (nonmetastatic) and 4C11+ (metastatic) were cultured in RPMI 1640 medium supplemented with 5% FBS and 1% penicillin (100U·mL−1) and streptomycin (100 μg·mL−1) at 37 °C in 5% CO2 humidified atmosphere. Cell culture reagents were purchased from PAN Biotech (Aidenbach, Germany). Axitinib (PZ0193; Sigma‐Aldrich, St. Louis, MO, USA), a selective inhibitor of VEGF receptors, and 5‐Aza‐2′‐deoxycytidine (5‐Aza‐CdR; Calbiochem, Merck, Darmstadt, Germany) were dissolved in DMSO (PAN Biotech) and stored at a final concentration of 10 mm at −20 °C. 4C11+ cells were treated with different concentrations (40 nm ‐10 μm ) of Axitinib for MTT assay and with 1 μm for all other assays. All treatments were performed for 48 h. 4C11− cells were treated with 10 μm of 5‐Aza‐CdR for 72 h. As a control, cells were treated with the respective volume of DMSO. Final DMSO volume in the cell culture was lower than 0.01%.
Dimethyl sulfoxide (dmso)
Manufactured by PAN Biotech
Sourced in Germany
DMSO is a clear, colorless, and odorless liquid that functions as a highly effective solvent. It is frequently used in various laboratory applications to dissolve a wide range of organic and inorganic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Axitinib, by Merck Group (1 mentions)
Dynabeads cd3, by Thermo Fisher Scientific (1 mentions)
Cd45 dynabeads, by Thermo Fisher Scientific (1 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
5 fluorouracil 5 fu, by Merck Group (1 mentions)
Thymoquinone tq, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Multiskan skyhigh microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Riluzole, by Merck Group (1 mentions)
Glutamate, by Merck Group (1 mentions)
Celltracker red and green, by Thermo Fisher Scientific (1 mentions)
Vybrant dio, by Thermo Fisher Scientific (1 mentions)
Vybrant dil, by Thermo Fisher Scientific (1 mentions)
6 protocols using dimethyl sulfoxide (dmso)
1
Murine Melanoma Cell Line Treatments
2
Immune Cell Activation with Dynabeads
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Dynabeads Human T-activator CD3/CD28, Dynabeads CD3, and Dynabeads CD45 (referred to as anti-CD3/anti-CD28, anti-CD3, and anti-CD45 beads, respectively) were purchased from Invitrogen Life Technologies (Carlsbad, CA). ML-7 was purchased from Merck Millipore (Billerica, MA) and suspended in dimethyl sulfoxide (DMSO) (Pan-Biotech, Aidenbach, Germany). Cells were preincubated with 30 µM ML-7 for 15 min and kept in the same concentration of the inhibitor throughout the experiment.
3
Preparation of 5-FU and Thymoquinone Solutions
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The single compounds, 5-fluorouracil (5-FU) and thymoquinone (TQ) were purchased from Sigma Aldrich. All investigated compounds were dissolved in 100% dimethyl sulfoxide (DMSO) (Pan Biotech) to 50–200 mM stock solutions, and stored at −20 °C. The stock solutions were further diluted in cell culture medium.
4
Metabolic Assay for FTC-133 and 8505C Cells
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To assess metabolic turnover of FTC-133 and 8505C cell lines, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Cat#M5655, Sigma-Aldrich) assay was performed. Cells were seeded in 96-well plates at a final density of 10000 cells/well/100 µL and incubated for 24 h. The day after, cells were exposed to increasing concentrations of FAC from 5 to 100 µM, and incubated for 24 h. MTT at a final concentration of 1 mg/mL was added to each well and incubated for 2 h and 30 min under standard culture conditions. Then, media were removed, 200 µL of MTT solvent, dimethyl sulfoxide (DMSO, Cat#P60-36720100, Pan-biotech) was added in each well, and plates were stirred on an orbital shaker for 10 min at room temperature. The absorbance was measured using a Multiskan SkyHigh Microplate spectrophotometer (Thermo Scientific) at 570 nm. Metabolic turnover was calculated as: (optical density sample/average optical density control)x100. Results were expressed as the percentage of MTT reduction versus control cells.
5
Pharmacological Evaluation of Neural Modulators
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6
Cell Labeling with CellTracker and Vybrant
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A 10 mM solution of CellTracker™ Red and Green (ThermoFischer) was prepared in sterile DMSO (PAN Biotech). H4-II-EC3 cells were incubated for 30 min in culture medium with 10 μM of CellTracker™ in the culture flask, before PBS washing and exposition to the TrypLE™ express enzyme. Alternatively, Jurkat and hMSCs were stained for Vybrant™ Dil (red) and PC-3 were labeled with Vybrant™ Dio (green), following the manufacturer instructions (ThermoFisher).
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