Cells were plated in 96-well plates with drugs at varying concentrations alone or in combination (Vel, Gilt, and Quiz alone for cytotoxicity assays, Vel and/or Gilt or Tal and/or Gilt for synergy assays). Plates were incubated for 72 h and were then terminated with CellTiter96 MTS Reagent (Promega) and incubated for an additional 2–4h for reagent metabolism. Absorbance was measured and used to determine the fraction of cells affected by treatment. Combination indices (if determining synergism) were calculated according to the Chou-Talalay method using CompuSyn software.
Celltiter96 mts reagent
Manufactured by Promega
Sourced in Australia
The CellTiter96 MTS Reagent is a colorimetric assay used to determine the number of viable cells in a sample. The reagent contains a tetrazolium compound that is reduced by metabolically active cells, resulting in the formation of a colored formazan product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid trolox, by Merck Group (1 mentions)
Sodium azide nan3, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Triethylamine, by Merck Group (1 mentions)
Proline, by Merck Group (1 mentions)
Syringaldehyde, by Merck Group (1 mentions)
Butylated hydroxyanisole bha, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Propargyl bromide, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
Prism 5, by GraphPad (1 mentions)
M1000 fluorimeter, by Tecan (1 mentions)
6 protocols using celltiter96 mts reagent
1
Cytotoxicity and Synergy Assays
2
Combination Drug Cytotoxicity Assay
3
Synthesis and Characterization of DES
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All chemicals were analytical grade and used without further purification. Sulfuric acid-hydrolyzed CNCs (98% freeze-dried powder, 0.8–0.9% S) were purchased from the University of Maine, USA. Ascorbic acid, butylated hydroxyanisole (BHA), copper (II) bromide (CuBr2), dimethylformamide (DMF), glycerol, proline, potassium carbonate (K2CO3), p-toluenesulfonyl chloride, sodium azide (NaN3), triethylamine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and syringaldehyde were purchased from Sigma-Aldrich. Propargyl bromide (80 wt% in toluene) was acquired from Acros Organics, and diethyl malonate and pyridine were from Tokyo Chemical Industries (TCI). 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was acquired from Bio-Strategy Pty Ltd, Australia. Dulbecco’s Modified Eagle Medium (DMEM) was purchased form Gibco™, and CellTiter 96® MTS reagent was purchased from Promega, Australia. Cream N (Neutrogena® Oil-Free Moisture) and Cream S (Neutrogena® Oil-Free Moisture Sun Protection Factor (SPF) 15,13.5% active ingredients) are commercially available facial creams purchased in a local pharmacy. DES was synthesized via proline-mediated Knoevenagel condensation of syringaldehyde and diethyl malonate (Figure S1 ), as we have reported previously [14 (link)].
4
Globo H Impacts Cell Viability
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The effect of Globo H on live
cells was measured with an assay for cell viability. MCF-7 cells (5000/well)
in medium were added to the wells of a 96-well plate from Corning
(Corning, NY) and grown for 3 days in the presence of Ac32FF (100 μM in 0.1% v/v DMSO),43 (link),44 (link) DMSO (0.1%
v/v) alone, or H2O2 (1 mM). The medium was replaced
with serum-free medium, and RNases in PBS were added at various concentrations.
Cells were then incubated for 44 h. The medium was removed, and the
cells were incubated in CellTiter 96 MTS reagent from Promega (Madison,
WI) for 2 h. The absorbance was then measured at 490 nm and normalized
to that from cells treated with 0.1% v/v DMSO alone (100%) and 1 mM
H2O2 (0%).
cells was measured with an assay for cell viability. MCF-7 cells (5000/well)
in medium were added to the wells of a 96-well plate from Corning
(Corning, NY) and grown for 3 days in the presence of Ac32FF (100 μM in 0.1% v/v DMSO),43 (link),44 (link) DMSO (0.1%
v/v) alone, or H2O2 (1 mM). The medium was replaced
with serum-free medium, and RNases in PBS were added at various concentrations.
Cells were then incubated for 44 h. The medium was removed, and the
cells were incubated in CellTiter 96 MTS reagent from Promega (Madison,
WI) for 2 h. The absorbance was then measured at 490 nm and normalized
to that from cells treated with 0.1% v/v DMSO alone (100%) and 1 mM
H2O2 (0%).
5
Cell Viability Assay with Tetrazolium Dye
6
Cell Viability Assay Protocol
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Cells were cultured in complete media (described above) in the absence or presence of the respective Cmax concentration determined for each respective drug dose curve. Cells were seeded at a concentration of 1.0 × 104 cells per well in a 96-well plate. Culture plates were kept in incubation at 37 °C, 5 % CO2 for 72-h. After 72-h, CellTiter96 MTS Reagent (Promega) was used according to manufacture instructions. Absorbance was recorded at 490 nm using a 96-well plate reader.
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