ATP Bioluminomics™ Cell-Based Assays

Although ATP bioluminescence assays were the first assays to be developed and produced by HemoGenix®, as of January 2012, HemoGenix® has expanded its assay kit product line to include fluorescence and colorimetric/absorbance readouts of the same assays originally developed using bioluminomics™ technology. This allows the investigator to perform essentially the same assays using instrumentation that might be more readily available without having to purchase another piece of equipment.

There is no doubt that the bioluminescence signal detection system incorporated into HemoGenix® bioluminomics™ assays represents the most sensitive assay readout available. In fact, ATP bioluminescence is even more sensitive (and safer) than any radioactive cell-labeling technique. Fluorescence and colorimetric/absorbance assays provide lower assay sensitivity. On the other hand, either an absorbance or fluorescence plate reader is usually available and these assays are less expensive than ATP bioluminescence assays.

Performing any of the HemoGenix® ATP bioluminomics™ assays (e.g. LIVEGlo™, STEMpredict™, CAMEO™-96, HALO®, LUMENESC™, STEMGlo™, XVPrime-Glo™ and CLGlo™ ) is a fast and simple process. Most assay kits contain everything required to culture cells and measure the intracellular ATP (iATP) concentration by bioluminescence. Since STEMAssays™, XVPrime™ Assays and CLAssays™ can be used for numerous cell types, these assay platforms provides the flexibility to use the investigators own protocols, culture reagents and conditions. The exceptions are NeuroGlo™ Complete, which not only contains cryopreserved neural stem cells, but also specialized medium to grow and expand the cells and HepatoGlo™, which contains medium to maintain hepatocytes. 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

Performing a bioluminomics™ assay requires a plate luminometer. For most, this might be an investment. However, compared to a good microscope or a flow cytometer, a plate luminometer is a relatively inexpensive investment and provides you with quantitative data on assays that are standardized and can be validated. 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
Hemocytometer or electronic cell counter to determine cell concentration
Hemocytometer or flow cytometer to determine 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, and especially bioluminomics™ assays. This is because all bioluminomics™ assays incorporate an ATP bioluminescence signal detection system, the most sensitive of all non-radioactive detection systems. Even very small pipetting errors in cell suspension and culture reagent dispensing as well as performing the ATP standard curve, can lead to very large coefficients of variation (CVs). To reduce pipetting errors, all pipettes must be professionally calibrated. Single and multichannel electronic pipettes are recommended since they are self-calibrating. However, even these must be professionally calibrated once or twice a year.

With three exceptions (CAMEO™-96, STEMClone™ and XVPrime-Clone™), all ATP bioluminomics™ assays involve culturing cells either in suspension or attached to the plate growth surface. CAMEO™-96, STEMClone™ and XVPrime-Clone™ are all methylcellulose-based ATP bioluminomics™ assays and are described in more detail on the last tab of this webpage (click here).

Click image for larger view

Please carefully read the instruction manual that accompanies every assay kit. Manuals for many of the assays can also be downloaded from the downloads page. When performing a bioluminomics™ assay, always wear gloves. The presence of ATP on the skin can dramatically influence the assay results. Also set up the plate luminometer prior to performing any ATP assays. If there are any questions, please contact HemoGenix® and speak to one of our technical personnel BEFORE starting to use the assay kit.

Little training is required to perform any bioluminomics™ assay. When ordering the first kit, extra reagents are provided free of charge, to perform several ATP standard curves and controls 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 pictures. Always perform an ATP standard curve every day samples are to be processed. It is a prerequisite and essential part of the assay.

The culture incubation time will depend on the assay, the species and the cells being analyzed.

It is important to use the number of replicates stated in the instruction manual to perform statistics and deal with possible outliers. Do not perform fewer replicates to save reagents. This usually leads 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.

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. Fluorescence assays are also less expensive than ATP bioluminescence assays and do not include the standards and controls presence in bioluminomics™ assays. In addition, whereas ATP bioluminescence assays require only 10 minutes to obtain results, fluorescence assays need to be developed for 0.5 - 2 hours. 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 incorporate a customized Promega CellTiter-Fluor™ reagent to measure cell viability and proliferation. This reagent detects a protease activity that is only present 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). Please not also that unlike ATP bioluminescence and absorbance assays, these fluorescence assays are not dependent upon the metabolic integrity and activity of the cells.

The HemoGenix® fluorescence assays:

HemoFLUOR™ (HemoFLUOR™-96, HemoFLUOR™-96 PCA^EQ, HemoFLUOR™-96 PMT)
ImmunoFluor™ (ImmunoFluor™-96, ImmunoFluor™-MLC)
MSCFluor™
STEMFluor™
XVPrime-Fluor™
CLFluor™

are performed in essentially the same manner as an ATP bioluminescence assay, except that performing an ATP standard and controls is not necessary.

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, results that may not be statistically significant and increased assay costs.

Absorbance Cell-Based, Viability and Proliferation Assays

Colorimetric / absorbance assays usually represent the least sensitive of the instrument-based cell proliferation assays. However, an absorbance plate reader can usually be found in most laboratories. The only addition would be a 490nm absorbance filter. Absorbance assays are usually the least expensive of all cell viability and proliferation assays. Whereas, results from an ATP bioluminescence assay are available in just 10 minutes and those from a fluorescence assay in 0.5-2 hours, it can take up 4 hours to develop sufficient sensitivity for an absorbance assay.

The colorimetric / absorbance reagent present in:

HemoLIGHT™ (HemoLIGHT™-96, HemoLIGHT™-96 PCA^EQ, HemoLIGHT™-96 PMT)
ImmunoLight™ (ImmunoLIGHT™-96, ImmunoLight™-MLC)
MSCLight™
STEMLight™
XVPrime-Light™
CLLight™

is a customized Promega CellTiter 96® AQueous One reagent to measure cell viability and proliferation.

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 will 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.

Methylcellulose, Clonal Proliferation and/or Differentiation Assays

HemoGenix® divides its assays into proliferation and differentiation assays. The reason for this is to provide its customers the correct assay for a specific application. If the goal of an experiment is to measure cell proliferation or a step involved in the proliferation process, a cell differentiation assay should not be used and visa versa. For example, there is no point in using a cell differentiation assay, such as the hematopoietic colony-forming cell (CFC) or unit (CFU) assay, to detect the proliferation ability or potential of hematopoietic stem cells. Similarly, detecting a cell membrane antigen by flow cytometry that is part of the differentiation process will not provide any information regarding the ability of that cell population to proliferate.

Although the processes of proliferation and differentiation overlap, from an assay viewpoint, they cannot be measured using the same assay readout. Either separate assays or different readouts of the same cells are required. Understanding this principle is important when drawing conclusions and interpreting data from experiments.

Differentiation assays include:

Molecular expression of genes and their products using, for example, PCR technology.
Intracellular expression of proteins using flow cytometry, ELISPOT etc.
Extracellular expression of proteins using flow cytometry.
Colony assays to detect differentiation ability and/or potential.

All HemoGenix® assays can be easily multiplexed to include assays in the groups 1-3 above. In other words, any of the ATP bioluminecence, fluorescence or absorbance proliferation assays can be multiplexed to obtain cell differentiation data by increasing the number of sample replicates and using different readouts to obtain increased information on the same sample.

All HALO®, HemoFLUOR™ and HemoLIGHT™ assays were originally based on the "classic" methylcellulose, colony-forming cell (CFC) or unit (CFU) assay, first described in 1966. In 1982, Rich and Kubanek described the first miniaturized methylcellulose CFC assay, which is now called CAMEO™-4 and comes in two versions, one for stem cell, basic and veterinary research and the other for stem cell processing laboratories. These are hematopoietic differentiation assays because the assay relies on the ability of cells to produce colonies of cells which can be identified, thereby identifying the cell population from which the colony originated. Although proliferation and division are required for the cells to produce the colony, the assay does not measure proliferation, but rather differentiation ability and/or potential.

The miniaturized CFC/CFU assay (CAMEO™-4) was the forerunner to what is now CAMEO™-96 (originally called HALO®-96 MeC). CAMEO™-96 is the methylcellulose version of the HALO® Platform using ATP bioluminomics™ technology to measure the proliferation of cells making up hematopoietic colonies after stimulation with cocktails of growth factors. However, since CAMEO™-96 is a methylcellulose assay allowing cells to grow under clonal conditions, it can also be used to measure two different readouts. Once the cells have been incubated, the colonies produced can be counted and differentiated directly in the wells on a 96-well plate. CAMEO™-96 is therefore a 96-well plate CFC/CFU assay. After counting the colonies, cell proliferation can then be measured using bioluminomics™ technology. This makes CAMEO™-96 quite unique in that it can measure both proliferation and differentiation using the same sample. But since measurement of colony proliferation using ATP bioluminescence requires an ATP standard curve to be perform, CAMEO™-96 is the only assay that allows true standardization of the CFC/CFU assay because total colony counts can be interpolated into ATP concentration equivalents.

This same procedure has been employed in two other assays, namely:

STEMClone™ and
XVPrime-Clone™.

Stem cells or ex vivo primary explanted cells with proliferation capability can be cultured and grown clonally in methylcellulose to detect and measure both proliferation and differentiation of the cells.

A note on dispensing methylcellulose reagents in CAMEO™-4, CAMEO™-96, STEMClone™ and XVPrime-Clone™ assays. Methylcellulose is a water-soluble, semi-solid viscous medium. As a result, methylcellulose is notoriously difficult to dispense accurately. Large variations between replicates are not uncommon. When using these assays, never dispense the methylcellulose Master Mix using a syringe and needle. This is a recipe for disaster. 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.