ATP Content in Cell Models

ATP Content in Cell Models2019-09-14T18:14:43-05:00

Background

  • Adensoine triphosphate (ATP) is the organic chemical that cells use to transport the energy they need to drive metabolic processes.
  • Measurement of total ATP content is useful as an indicator of the overall viability of cell models
  • ATP is useful in the context of plate reader based assays to calculate IC50 values, or to identify potentially toxic compounds in a high throughput manner.
  • Using Celltiter-Glo, ATP content can be assessed in 3D cell models to evaluate overall viability of cells within the model.
  • This assay can also be conducted on adherent monolayers of cells.
  • A related assay, Realtime-Glo, looks at the metabolic redox potential of cells. Realtime-Glo is useful as it is not an endpoint assay, allowing for the monitoring of cell model viability multiplexed with imaging based endpoints. Using Realtime-Glo, counter screens for toxicity can be conducted on the same disease models being tested for compound efficacy, or be used to triage models for more in depth imaging for mechanisms of cell death or dysfunction.

Protocol

InstrumentThermoFisher Varioskan
Analysis MethodPlate Reading Luminometer
MarkersLuminescence based Viability
Luminescence based Metabolic Potential
Cell Types AvailableATCC cell lines (e.g. HepG2, A549, BT549, MCF-7, and others)
Other cell lines available on request
Test Article Concentration8 point assay (0.05, 0.1, 0.5, 1, 5, 10, 50, 100 µM)
(custom concentrations available)
Number of Replicates3 replicates per concentration
Quality Controls0.5% DMSO (vehicle control)
Staurosporine (positive control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryDose response curves, IC50 values

General Procedure

  1. Tumor spheroids grown by seeding 1000 cells/well into ULA U-bottom plates
  2. Tumor spheroids grown to approximately 200 μm in diameter
  3. Treatment with test compounds
  4. After 24 hours, tumors are lysed with Celltiter-Glo
  5. Luminescence is measured on a plate reader

Multiplexing Alternative

  1. Tumor spheroids grown by seeding 1000 cells/well into ULA U-bottom plates
  2. Tumor spheroids grown to approximately 200 μm in diameter
  3. Treatment with test compounds and Realtime-Glo
  4. Viability of the model is monitored using luminescence
  5. Spheroids are fixed and labeled with relevant imunofluorescent labels
  6. Spheroids are cleared and imaged
  7. Images are analyzed
This website uses cookies to enhance the user experience. Ok