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Background - Performing a Bioluminonics Assay
  
 
  • ATP Bioluminescence

    Assays

Background

Standardized ATP Bioluminescence Assays

What are ATP Bioluminescence Assays?

HemoGenix® ATP bioluminescence assays are standardized measurments of the cell's energy source in the form of ATP (adenosine triphosphate) to provide information on:

  • Metabolic viability (cellular and mitochondrial integrity)
  • Cellular functionality
  • Cell "quality" or proliferation ability
  • Cell potency or proliferation potential
  • Cytotoxicity
  • Cell number
  • Apoptotic status.

All HemoGenix® ATP bioluminescence assays promote "Best Practice Criteria Testing". The readout is the standardized and validated measurment of changes in intracellular ATP (iATP) concentration when it reacts with luciferin and luciferase to produce bioluminescence in the form of light. The light is measured as a "glow" in a luminescence plate reader. The information obtained from all bioluminescence assays is quantitative and standardized allowing the assay to be validated according to regulatory guidelines.

The Concept of Measuring Bioluminescence 

 

The Concept of Measuring ATP Bioluminescence

 

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ATP is used as a biochemical marker for multiple readouts. This is because changes in the iATP concentration as a result of cellular and mitochondrial activity directly correlate with cell proliferation, inhibition, cell number and even apoptosis. ATP is therefore an excellent, quantitative marker of cell viability.

For most ATP bioluminescence assay applications, a simple luminescence plate reader can be used such as the Berthold SpectroLia. For more complex and high throughput applications, including using 384-well plates, a Molecular Devices SpectraMax L with SoftMax Pro software is recommended. 

 

ATP Bioluminescence Cell-Based Assays

ATP bioluminescence assays provide the most sensitive readout of any signal detection system, including radioactive readouts. 

Performing any HemoGenix® ATP bioluminescence assay (e.g. LIVEGlo™, CAMEO™-96, HALO®, ImmunoGlo™, MSCGlo™, STEMGlo™ ) is a fast and simple process. Most assay kits contain everything required to culture cells and measure the intracellular ATP (iATP) concentration. Some assays even include a vial of frozen cells. Most assay kits contains the following components:

  • Culture Master Mix.
  • ATP standard
  • ATP high and low controls
  • Sterile, 96- or 384-well culture plates
  • Non-sterile 96- or 384-well plates for the ATP standard curve and controls
  • ATP Enumeration Reagent
  • Sterile, adhesive foils for keeping unused wells sterile
  • Instruction manual

Equipment and Supplies to Perform Bioluminescence, Fluorescence or Absorbance Assays

Compared to a microscope, automated colony reader or flow cytometer, a plate reader is usually an inexpensive investment that provides quantitative data for many other HemoGenix® products In addition to the instrument, the following general laboratory equipment is required:

  • Laminar flow biohazard hood
  • Sterile plastic tubes (5ml, 10-12ml, 50ml)
  • Single channel pipettes (1µl to 1,000µl) preferably electronic (motorized) since these are self-calibrating
  • 8- or 12-channel pipette (10µl to 100µl), preferably electronic (motorized) since these are self-calibrating
  • Reservoir for multi-channel pipette
  • Sterile and non-sterile pipette tips
  • Automated cell counter to determine cell concentration and viability
  • Humidified, tissue culture incubator at 37°C with 5% CO2 (minimum requirement) and 5% O2 (preferable)
  • A liquid handler should be used for all 384-well plate assays

Good pipetting technique is essential for all microassays. Small errors in pipetting results in large coefficients of variation (CVs) regardless of the readout used. All pipettes should be professionally calibrated at least once a year.

Step-by-step process of performing an ATP bioluminescence assay

 

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Please carefully read the assay manual that accompanies every assay kit. Manuals for many of the assays can also be downloaded from the downloads page. For any assay, especially ATP bioluminescence assays, always wear gloves. The presence of ATP on the skin can dramatically influence the assay results. In addition, set up the plate reader prior to performing any assay. For ATP bioluminescence assays, an ATP standard curve is required prior to measuring any sample. If there are any questions, please contact HemoGenix® and speak to one of our technical personnel BEFORE starting to use the assay kit.
 

All HemoGenix® instrument-based assays can be learnt within 1-2 days. For ATP bioluminescence assays, extra reagents are provided free of charge for the first assay kit, to perform several ATP standard curves in order to achieve the best dispensing and mixing technique. Additional Instrument Optimization Reagents, ATP standards and high and low controls are also available. The essential steps in performing any bioluminomics™ assay are shown in the diagram opposite.

For any instrument-based assay, it is important to perform the number of replicates stated in the assay manual. Reducing the number of recommended measurements may save reagents, but could lead to inconclusive results and more expense. In addition, please do not use reagents and supplies, e.g. plates, that are not included with the assay kits. All components are optimized to provide the highest quality results. Changing any of the components will void the assay kit warranty. 

 

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