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Assays: Wellular to Cellular

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Individual stars in the sky to illustrate the individual cells possible

Out with the Old…

Well-based assays have been the standard for common laboratory experiments, such as fluorescence cytometry. A researcher places a small amount of sample into a well on a plate and assays it, which produces a single data point. However, this so-called single data point is actually an average of the measurements of each cell within the sample.

Importantly, the sample could generate a much larger set of data points if each cell was sampled individually.

There are other limitations to using well-based assays:

  • Non-physiological: Because individual cells cannot be identified, important data is missed. For example, if sub-populations within a well vary in response or behavior, then that data is not captured.
  • Chemical characteristics cannot be determined: Well-based assays treat the sample as one entity, as such cell-specific characteristics cannot be determined. For example, in drug testing, beyond overall viability, knowing other outcomes (such as membrane permeability) is very important. These other outcomes are indistinguishable with well-based assays.
  • Sticky cells must be enzymatically detached: This subjects all the cells in the sample to wash steps with harsh chemical treatments to quantify adherent cells.

…In with the New (Image Cytometry)!

Image cytometry multiplies the applications of traditional flow cytometry by adding high-resolution visualization and acquisition of single cells through the use of a microscope. This allows assaying of each, individual cell. Image cytometry results in a higher degree of cell-counting accuracy and enables researchers to observe the heterogeneity within cells in a sample.

This method of cell quantification and visualization has multiple advantages over traditional techniques, such as general fluorescence cytometry or flow cytometry.

Reveal the Distribution of Cells

Heterogeneity in a “wellular” sample is completely missed by traditional plate assays, because it only acquires a single data point that averages the data for all cells in the sample. This method supplies critical information about the different populations of cells present in the sample by acquiring data for each individual cell. It can be very useful when describing how cells respond to a treatment, or whether cells are distributed evenly in the well.

Quantifies Adherent Cells Directly

Cells that are “stuck” together cannot be counted by traditional flow cytometry without first undergoing physical and chemical separation. These extra processing steps are often damaging to cells and take up valuable time. Using real-time image cytometry, researchers can automate the cell counting of adherent cells while bypassing these extra steps.

Protects Cells

This method protects cell by offering different imaging modes to visualize cells instead of introducing dyes. Fluorescent assays involve the addition of toxic reagents to produce a measurable signal. Rather than introducing toxic reagents to intercalate DNA, determine cell mass, or assay cell proliferation, the combination of microscopy with cytometry allows visualization of cells through modifying the imaging mode. For example, clear images can be obtained using phase contrast and bright-field.

Miniaturization of Assays

As described above, image cytometry negates the need for physical and chemical processing of cells for assays. As a result, fewer cells will be lost in preparation, which allows researchers to use a smaller starting volume. This reduces both sample and reagent costs.

Real-Time Image Cytometry

As a result of the many applications and benefits over standard techniques, image cytometry has become a household tool for many laboratories. Technological advances to improve the quality and automation of data acquisition has resulted in innovative additions to image cytometry resulting in a next-level upgrade. Now, single-cell images can be acquired so rapidly that experiments can be visualized in real time to reveal rates of apoptosis, proliferation, and more.

The ability to capture dynamic events during the assay allows the full range of cellular behavior to be observed and quantified. Throughput is dramatically increased as data from every single cell is collected throughout the course of the experiment. As a result, no critical events are missed. Images of interest can also be captured and saved for future applications.

Many well-based assays can benefit from the throughput multiplying power of real-time imaging. Other useful applications include cell viability/toxicity assays, cell migration assays, and cell senescence assays.

Real-Time Solutions

Real-time image cytometry emerges as a powerful upgrade to standard well-based assays, improving the detection and increasing the amount of information that can be obtained from samples. However, in a world where square-footage is a valuable commodity, increasing your lab footprint with an imager and plate reader for cell-based assays is a viable concern. That’s why eco-conscious real-time image cytometers are the technology of the future, combining the microscope with the plate reader in one powerful instrument.

Another concern among laboratories is that real-time assays with live cells can take a long time when compared to fixed samples. The experiment could take days, and continuous monitoring by researchers can become a strain. The best solution to this is using an automated workflow that has a real-time, easily accessed monitoring system, like CNS by Tecan. This solution keeps track of the experiment so that you don’t have to and allows you to perform actions remotely. This important feature means that you can stop the experiment at any point if it doesn’t proceed as expected, add reagents at specific times during the workflow, and record specific clusters of data.

By increasing experimental power and control, the combination of microscope-integrated cytometry + real-time imaging + real-time monitoring delivers a breakthrough in improving the productivity and quality of cell-based assays.

Image Credit: Tom Hall

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