The essential cell analysis tool

New designs in flow cytometers have extended the use of multi-parametric assays from the core labs onto the bench space of life science research laboratories. Mainstream cell biology applications can now be addressed with a single platform.

“New designs in flow cytometers have extended the use of multi-parametric assays from the core labs onto the bench space of life science research laboratories. Mainstream cell biology applications can now be addressed with a single platform”

Today's life science research conundrum

Today's life science researchers utilize technologies from many disciplines, such as molecular biology, immunology and cell biology, to advance their research. Purchasing, and then mastering varied instruments and technologies devours time and monetary resources. Often a technique may be needed in only a single experimental phase leading to an accumulation of infrequently used instruments cluttering valuable bench space. The ideal solution is an instrument, which can perform a myriad of multi-parametric applications.

With a footprint, weight and price similar to a real-time qPCR system or microplate reader, the Accuri C6 Flow Cytometer® System puts an essential cell-analysis tool into the hands of more researchers than ever before. The C6 offers all the performance features of larger, complex flow cytometers and, combined with the straightforward CFlow® software, does not require expert operators, making flow cytometry equally available to both novices and experts.

The issues

As with so many technologies, over time, manufacturers have added a seemingly endless series of bells and whistles to mature flow cytometric platforms, rendering them increasingly cumbersome. Only trained, experienced operators can effectively use these systems and workloads can back up to the point where progress is gated by access to a core lab.

Due to the wide variations in fluorescence and light scatter signals produced by different types of cells and particles, flow cytometrists often spend significant time and valuable sample, optimizing amplifier gains and/or PMT voltage settings. These settings cannot be changed post-analysis if the cytometrist determines that signals were improperly amplified, resulting in the irretrievable loss of data, even on instruments using digital signal processing.

The Accuri C6 Flow Cytometer (change link to http://www.AccuriCytometers.com/products) is equipped with pre-optimized detectors calibrated to operate within their linear range, so it can be used to analyze a wide variety of samples, ranging from dim, barely fluorescent, micron-sized platelets through large, >30 micron, highly fluorescent cell lines. The combination of high-resolution digital-signal processing (24-bit DSP), resulting in an expansive dynamic range on all detectors, and the CFlow software capability to "zoom" in on very small areas of data display make the C6 easy to use for both novices and experienced flow cytometrists.

Gating strategies and fluorescence compensation values can be set during, or after, data collection. Data collected on a C6 is always retained and can be re-analyzed, at any time, if gating or compensation errors are discovered, or in light of new research findings

Varied Biological Applications

Microscopy, microplate reading, qPCR and Western blots are frequently used for a variety of cell studies. The Accuri C6 Flow Cytometer offers several unique advantages by enabling multi-parametric individual cell analysis. The C6 can analyze single to hundreds of thousands of individual cells, from heterogeneous populations and flag rare events involving just a few cells. Another powerful feature is the C6's ability to quantitatively measure the concentration of cells or particles in samples. Absolute cell counts are correlated with the specific, known volumes being sampled.

We will now examine how the C6 can improve outcomes from some existing techniques.

Western Blots

Although Western blots are considered a gold standard for protein biomarker determination analysis is performed on entire cell populations instead of individual cells, often making it difficult to identify phenotypes in small subsets of the population. For example, when assessing apoptosis-related protein expression, flow cytometry offers specific advantages. Flow cytometry can characterize the aberrant disease-associated phenotypes of viable Minimal Residual Disease (MRD) associated cells. Flow cytometric analysis is rapid, highly sensitive, selective and allows quantitation. Western blots simply cannot address the low levels of MRD associated cells in experiments with apoptosis-resistant cells.

GFP Transfection Studies

Flow cytometry can accurately quantitate reporter gene expression (Green Fluorescent Protein (GFP) for example) in each cell in a transfected population. Co-transfection of a reporter plasmid and a reference plasmid can be quite variable in normal human cells, making interpretation difficult in reporter gene assays. A flow cytometry can quantitate both reporter and reference plasmid expression at the single cell level, even in cases of low transfection efficiency. As opposed to bulk detection approaches examining the whole well in a microplate, flow cytometry analysis of individual viable cells can pick up very rare events and can identify rare subpopulations.

Continuous measurement of intracellular Ca²⁺

An alteration in intracellular calcium ions (Ca²⁺) is one of the most rapid cellular responses to a variety of stimuli, yet obtaining accurate data on the dynamics of intracellular calcium flux is a major challenge. Specialized liquid handling and fluorescence microplate reading systems have been used to examine cellular bulk population behavior during investigations of the rapid responses of intracellular Ca²⁺ to various stimuli in vitro. The Accuri C6 Flow Cytometer, which operates with an open, as opposed to a pressurized, fluidics system allows addition of agonists, to study the effects of modulators on Ca²⁺ in mixed populations, with continuous recording, providing a method for highly accurate, dynamic calcium measurements. With conventional flow cytometry, the run has to be halted, the sample tube opened and agonist added, then the tube resealed to recommence data acquisition, which adds a gap, or blind spot, in the data.

Cell cycle analysis and ploidy

Plant nuclear DNA content (ploidy) varies over extreme ranges and quantitative measurement of the characteristic "C-value" is achieved by detecting propidium iodide fluorescence using flow cytometry. In typical flow cytometers, the Nuclear DNA measurements are often hampered by excessive cellular and sub-cellular debris and autofluorescence from other prevalent cellular components, such as chloroplasts. The signal range is filled before enough data points for the nuclei of interest can be collected. With over 6 decades of digital signal linearity, the Accuri C6 Flow Cytometer is the first flow cytometer to cover the full biological range of flowering plant genome sizes from 0.32 to 80.9 picograms in a single run.

The Future of Flow Cytometry

Multi-parametric flow cytometric analysis can now replace other less accurate techniques and be accomplished as experimental needs dictate. By designing a 2-laser, 6-detector, compact flow cytometer, Accuri has successfully addressed the mainstream cell biology applications with a single platform and freed up valuable resources.

Download the full-length white paper to at www.AccuriCytometers.com/welcome/cell-analysis.