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Background - Performing a Bioluminonics Assay
  
 
  • ATP Bioluminescence

    Assays

  • Fluorescence Assays

  • Absorbance Assays

  • Methylcellulose

    Clonal 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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Fluorescence Cell-Based Viability and Proliferation Assays

 

Although fluorescence assays are less sensitive (10 - 100 times) than ATP bioluminescence assays, they represent a middle ground for performing cell-based proliferation assays and are less expensive. A 0.5 - 2 hour signal development time is required for fluorescence assays. To measure fluorescence, a plate luminometer is required with an excitation filter of 390-400nm and an emission filter at 505nm.

All HemoGenix® fluorescence assays uses a customized reagent that detect a protease activity present only in live cells. The reagent contains a glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) which permeates the cells and is cleaved by live-cell protease activity to produce a fluorescence signal detected at 505nm. The amount of fluorescence is proportional to the number of cells and therefore not only detects cell viability, but also cell proliferation ability. If the protease leaks into the extracellular medium due to the loss of membrane integrity, it becomes inactive. Note that loss of membrane integrity can indicate a loss in cell viability. (This should be compared to dye exclusion viability methods). 

HemoGenix® fluorescence assays include:

  • HemoFLUOR™ (HemoFLUOR™-96 Research, HemoFLUOR™-96 PCAEQ, HemoFLUOR™-96 PMT, HemoFLUOR™-96 TE)
  • ImmunoFluor™ (ImmunoFluor™-96, ImmunoFluor™-MLC)
  • MSCFluor™-96 Research
  • STEMFluor™-96 Research

These assays are performed in essentially the same manner as an ATP bioluminescence assay, except that no standard and controls are required.

If Master Mixes are provided with the assays, they are dispensed into separate tubes for each sample to be measured. The cell suspension being tested is adjusted to the correct working concentration and added to the Master Mix. The contents are mixed together and replicate wells dispensed into the 96-well plates provided with the kit. After the required cell incubation time has elapsed, 0.1ml of the fluorescence reagent included with the kit is added to each well, mixed and further incubated for 0.5 - 2 hours. The plate can be removed from the incubator at any time to measure fluorescence at 505nm and then returned to the incubator if need be. It is not necessary to use the whole 96-well plate at once. 

If culture master mixes are not included with the kit, this provides the investigator the flexibility to use their own reagents, protocols and culture conditions. It is recommended to prepare the culture reagents as a Master Mix first, prior to preparing and adjusting the cell concentration.

An important aspect of any assay is to obtain statistically signficant results. For all fluorescence assays, a minimum of 6 replicate wells is recommended. Fewer replicates can be performed, but this may lead to the inability to remove outliers. Performing fewer replicates may lead to results that may not be statistically significant and increased assay costs.

Absorbance Cell-Based, Viability and Proliferation Assays

 

Although colorimetric / absorbance assays usually represent the least sensitive of the instrument-based cell proliferation assays, they are also the most cost-effective and an absorbance plate reader can usually be found in most laboratories. The only addition would be a 490nm absorbance filter. Absorbance assays produce a color reaction that can take up 4 hours to develop. Optimal color development takes about 3 hours and the culture plate can be removed and replaced into the incubator at any time to monitor the reaction.

 

Colorimetric / absorbance assays include:

  • HemoLIGHT™ (HemoLIGHT™-96 Research, HemoLIGHT™-96 PCAEQ, HemoLIGHT™-96 PMT, HemoLIGHT™-96 TE)
  • ImmunoLight™ (ImmunoLIGHT™-96, ImmunoLight™-MLC)
  • MSCLight™-96 Research
  • STEMLight™-96 Research.

 

Many absorbance viability assays use an MTT terozolium substrate which forms an insoluble yellow formazan product, which then requires the addition of a second reagent to solublize the formazan product so that it can be read in the instrument. In contrast, all HemoGenix® absorbance assays incorporate a novel tetrazolium compound called MTS. In the presence of phenazine ethosulphate (PES), an electron coupling reagent, the MTS is reduced to a soluble formzan reagent, thus obviating the need for a second step prior to measurement. The reaction requires the presence of NADPH or NADP produced by dehydrogenase enzymes to accomplish the terazolium reduction step. As a result, the MTS absorbance assay detects metabolic integrity and activity and is therefore a metabolic viability assay.

 

When cells are stimulated to proliferate, metabolic activity increases several fold. The amount of absorbance produced as a result of increased MTS reduction therefore correlates with increased proliferation activity and can thus be used as a proliferation assay. 

 

All HemoGenix® absorbance assays are performed in the same manner as ATP bioluminescence and fluorescence assays. Like the fluorescence assays, the absorbance assays to not include a standard and controls. However, the assay kits do include a non-sterile, 96-well plate to perform a background absorbance reading, which is subtracted from the sample absorbance measurement.

 

Depending on the cell type being detected, high cell concentrations may be necessary to obtain significant results. It is recommended to perform a cell dose response in order to obtain the optimal cell concentration used for the assay. As with ATP bioluminescence and fluorescence assays, it is also necessary to use a sufficient number of replicate wells in order to obtain statistically significant results. Six replicates are recommended, since by using fewer replicate wells, especially when fresh or cryopreserved primary cells are employed, problems might occur that would require the assay to be repeated, thereby involving increased costs.

Clonal Methylcellulose Assays - Colony-Forming Assays

Methylcellulose is a water-soluble, viscous, inert substance that can be used in place of agar and other semi-solid media, to grow cells under clonal conditions. It should be emphasized that although colony growth requires proliferation, the latter cannot be quantitatively measured using a clonal assay. Virtually all proliferating cells can be grown under clonal conditions to produce colonies.

Hematopoietic cells are often grown in culture under clonal conditions as colony-forming cells (CFC) or units (CFU). These assays detect differentiation ability because the type of colony produced is dependent upon the cells differentiating and maturing so that the mature cells will identify the type of colony and the cells from which it is produced. 

All HALO®, HemoFLUOR™ and HemoLIGHT™ assays were originally based on the "classic" methylcellulose, CFU/CFC assay. HemoGenix® has 4 different methylcellulose assays available:

  • ColonyGro™: A "classic" 12-14 day methylcellulose culture reagent (100ml) for hematopoietic cells grown in 35mm Petri dishes. The 100mL volume of ColonyGro™ is sufficient for 44 samples, compared to 30-33 samples from competitors.
  • CAMEO-4™: A miniaturized CFU/CFC assay kit that includes 50 culture plates to measure 50 samples in 4 x 0.1ml cultures. Colonies can be counted after 9-10 days.
  • CAMEO™-96: An ATP bioluminescence proliferation assay combined with a CFC/CFU methylcellulose differentiation assay for hematopoietic cells performed in a 96-well plate. Colonies can be counted between 7 and 9 days.
  • STEMClone™: Similar to CAMEO™-96, but allows the user to include their own reagents a growth factors.

Standardized Reagents vs Standardized Assays

Standardized reagents are manufactured under standardized conditions, usually Good Manufacturing Practices or GMP. Standardized reagents does not mean that the assay is standardized. An assay can only be standardized if external standards and controls are included. With the exception of CAMEO™-96, none of the clonal, methylcellulose assays are standardized assays. This is contrary to what some companies may want you to believe.

A Note on Dispensing Methylcellulose

Methylcellulose is notoriously difficult to dispense accurately. Large variations between replicates are not uncommon. When using these assays, never dispense the methylcellulose reagent using a syringe and needle. This is a recipe for pipetting error. Please always use a positive displacement manual or electronic (motorized) pipette to dispense the methylcellulose reagent. This will produce lower pipetting errors with decreased variability.