To determine the effect of transient RAN knockdown on cell proliferation, the RT-CES system, as described (Diskin et al., 2012 (link)), was utilized, and cell viability was measured using CellTiter- Glo Cell viability assays (Promega) following the manufacturer’s protocol, with further details described in the Supplemental Experimental Procedures .
Celltiter glo cell viability assay
Manufactured by Promega
Sourced in United States, United Kingdom
The CellTiter-Glo cell viability assay is a luminescent-based method for quantifying the amount of adenosine triphosphate (ATP) present in a cell population. It provides a simple and sensitive way to measure the metabolic activity of cells, which is indicative of their viability.
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Lab products found in correlation
Mts assay, by Promega (4 mentions)
Trypan blue solution, by Merck Group (4 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (4 mentions)
Lumistar microplate luminometer, by BMG Labtech (3 mentions)
Envision 2103, by PerkinElmer (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Synergy 4 microplate reader, by Agilent Technologies (2 mentions)
Bright glo luciferase assay, by Promega (2 mentions)
Omg fluorostar plate reader, by BMG Labtech (2 mentions)
Celltiter glo 3d cell viability assay, by Promega (2 mentions)
Doxycycline, by Takara Bio (2 mentions)
Plx4720, by Selleck Chemicals (2 mentions)
Human phospho rtk array kit, by R&D Systems (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
P rtk, by R&D Systems (2 mentions)
Sirna against mitf and non targeting control sirna, by Horizon Discovery (2 mentions)
Hb egf, by R&D Systems (2 mentions)
Azd6244, by Selleck Chemicals (2 mentions)
U0126, by Selleck Chemicals (2 mentions)
Vemurafenib, by Selleck Chemicals (2 mentions)
118 protocols using celltiter glo cell viability assay
1
Transient RAN Knockdown Impacts Proliferation
2
Cytotoxicity and CYP3A4 Enzymatic Assays
3
Cellular Viability and Proliferation Assays
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For MTT assay, 8000 cells per well were plated on a 96‐well plate. After indicated treatment, 100 µL fresh medium containing 10 µL of 5 mg·mL−1 MTT (Sigma‐Aldrich) was added and incubated at 37 °C for 2 h. DMSO was used to dissolve the crystals formed, and the absorbance was measured at 570 nm through a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA).
For CellTiter‐Glo cell viability assays (Promega, Madison, WI, USA), 2000 cells per well were plated in a 96‐well plate. After the indicated treatment, the CellTiter‐Glo reagent was added according to the manufacturer's instructions, and the luminescent signal was measured using Synergy H4 microplate reader (BioTek, Winooski, VT, USA).
For trypan blue exclusion assay, 10 000 cells per well were plated on a 24‐well format, and after being treated with indicated conditions, they were trypsinized and stained with 0.4% trypan blue solution (Corning, Glendale, AZ, USA). The cell number was then counted with a hemocytometer.
For colony formation assays, 500 cells per well were seeded on a six‐well plate and ARN‐3236 was added at the indicated concentrations. After 10–12 days, the cells were fixed and stained with Giemsa solution (Merck, Kenilworth, NJ, USA) containing 50% methanol. Colonies were then manually counted.
For CellTiter‐Glo cell viability assays (Promega, Madison, WI, USA), 2000 cells per well were plated in a 96‐well plate. After the indicated treatment, the CellTiter‐Glo reagent was added according to the manufacturer's instructions, and the luminescent signal was measured using Synergy H4 microplate reader (BioTek, Winooski, VT, USA).
For trypan blue exclusion assay, 10 000 cells per well were plated on a 24‐well format, and after being treated with indicated conditions, they were trypsinized and stained with 0.4% trypan blue solution (Corning, Glendale, AZ, USA). The cell number was then counted with a hemocytometer.
For colony formation assays, 500 cells per well were seeded on a six‐well plate and ARN‐3236 was added at the indicated concentrations. After 10–12 days, the cells were fixed and stained with Giemsa solution (Merck, Kenilworth, NJ, USA) containing 50% methanol. Colonies were then manually counted.
4
Cell Viability Assessment via CellTiter-Glo
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Cell viability was measured using CellTiter-Glo cell viability
assay (Promega G7573). Cells were cultured in 96-well plates and,
after transfection, were incubated for 24 h, the medium was removed
and 100 μL of the CellTiter-Glo reagent was added to each well
and mixed by gentle shaking. The plate was incubated at room temperature
for 10 min and the luminescence recorded using a plate reader (PerkinElmer,
2103 Envision).
assay (Promega G7573). Cells were cultured in 96-well plates and,
after transfection, were incubated for 24 h, the medium was removed
and 100 μL of the CellTiter-Glo reagent was added to each well
and mixed by gentle shaking. The plate was incubated at room temperature
for 10 min and the luminescence recorded using a plate reader (PerkinElmer,
2103 Envision).
5
High-Throughput Cell Viability Assay
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The activity levels of single agents and combinations were determined by a high-throughput CellTiter-Glo cell viability assay (Promega). Cells (1–2 × 103) were plated in each well of 384–well plates using a Precision XS liquid handling station (Bio-Tek Instruments, Winooski, VT) and incubated overnight. Drug source plates were prepared in 96-well Megatiter plates (Neptune Scientific, San Diego, CA), and the Precision XS station was used to transfer drugs to four replicate wells with an additional four control wells receiving DMSO vehicle control without drug. At the end of the drug incubation period, CellTiter-Glo or Caspase-Glo reagent was added to each well at 1:1 ratio (v/v) with media. The luminescence of the product of viable cells was measured with a Synergy 4 microplate reader (Bio-Tek Instruments). The luminescence data were transferred to Microsoft Excel to calculate percent viability. IC50 values were determined using a sigmoidal equilibrium model regression and XLfit version 5.2 (ID Business Solutions). The IC50 values obtained from single-drug cell viability assays were used to design subsequent drug combination experiments. High-throughput two-agent combination screening experiments were performed using a 5 × 5 matrix format in 384-well plates to interrogate 25 individual concentration ratios per combination (Supplemental Figure S1 ).
6
Cytotoxicity Assay for Ovarian Cancer Cells
7
Mammalian Cell Toxicity of Tartrolon D/E
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Example 11
Toxicity of the compounds of the invention for mammalian cells was determined. Bovine Turbinate (BT) cells (ATCC #CRL-1390) are a primary cell line (not a transformed cancer cell line) and were used to provide a preliminary evaluation of the toxicity of tartrolon D/E for mammalian cells. Briefly, BT cells were cultured in the presence of increasing starting concentrations of Tartrolon D/E for 24 hours. Cell proliferation was measured using CellTiter Glo Cell Viability Assay (Promega) and the data was analyzed with GraphPad Prism. The results showed that the toxic dose at which 50% of the cells were killed was 1.14 μM (
8
Cell Proliferation and Cell Cycle Analysis
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Cell proliferation was evaluated by using the CellTiter-Glo cell viability assay (Promega) following the manufacturer’s instructions. Briefly, 1500 cells/well were plated in 96-well plates in the presence of miRNAs, siRNA, or inhibitors. The plates were then incubated for 3–7 days, then 100 μL of CellTiter-Glo reagent was added to lyse the cells. After a 10-min incubation at room temperature, luminescence was recorded in a luminometer with an integration time of 1 s per well.
For cell cycle analysis, transfected cells were harvested at 48 h and fixed at 4 °C. Propidium iodide (50 μg/mL) was used to stain the fixed cells. DNA content was analyzed using a FACSCalibur instrument (Becton Dickinson Bioscience). Cell cycle fractions were quantified with Multicycle Software (Phoenix Flow Systems) and analyzed by FlowJo software.
For cell cycle analysis, transfected cells were harvested at 48 h and fixed at 4 °C. Propidium iodide (50 μg/mL) was used to stain the fixed cells. DNA content was analyzed using a FACSCalibur instrument (Becton Dickinson Bioscience). Cell cycle fractions were quantified with Multicycle Software (Phoenix Flow Systems) and analyzed by FlowJo software.
9
ATP-Based Cell Viability Quantification
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According to the manufacturer, Promega Corporation, the CellTiter-Glo® cell viability assay detects live cells by quantifying the amount of ATP present in living cells. The reagent quickly lyses the cells, stabilizes the ATP present, and generates a luminescent signal proportional to the amount of ATP present. The signal is directly proportional to the number of living cells in the sample. The test was performed according to the manufacturer's instructions and those contained in the package insert of the kit.
10
Cell Proliferation Assay with trtE
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HCT8, HFF and BT cells were seeded in 96-well plates to achieve 20–30% confluence after 24 hours of growth. Dilutions of trtE or DMSO were added to the wells and proliferation of the cells evaluated 24 hours later by quantifying cellular ATP levels with CellTiter-Glo Cell viability assay (Promega).
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