BD Accuri™ C6 Plus Flow Cytometer

Data is acquired from the Collect tab. All tasks and settings are laid out simply on one screen for quick access and operation. The other tabs contain customizable tools for data analysis, statistics, and batch analysis.

Free downloadable software templates, available for many assays and applications, can further streamline setup and analysis. BD Accuri C6 Plus software files can be exported in FCS 3.1 format for seamless data import into flow cytometry analysis programs such as FCS Express™ software and FlowJo™ software.

The unique fluidics system of BD Accuri flow cytometers, driven by peristaltic pumps, allows them to determine sample volume, as well as count cells, directly. This feature speeds and simplifies cell analysis by calculating cell concentrations (per unit sample volume) automatically. Accurate cell concentrations are essential in many research applications, including enumerating leucocytes, B cells, T cells, and platelets in human blood, measuring microorganism concentrations in purified water, and determining the viability of cultured cell lines. Direct counts on BD Accuri systems correlate highly with counting beads, and are more precise than hemocytometer counts.

You can download two BD Biosciences documents on cell counting from the list below. The technical bulletin contains recommendations, tips, techniques, and troubleshooting suggestions to help maximize the accuracy of cell counts and concentrations on BD Accuri systems. The white paper presents sample data and guidance for viable cell concentrations in cultured cell lines, immune cell concentrations in human peripheral blood, and platelet counts in whole, unlysed human blood.

Resources

• Technical Bulletin: A Guide to Absolute Counting Using the BD Accuri™ Flow Cytometer
• White Paper: Determining Cell Concentration by Direct Volume on the BD Accuri™ Flow Cytometer
• Webinar Slide Set: Cell and Platelet Counting Using the BD Accuri™ C6 Flow Cytometer

Sample Data

Cytokines and growth factors are a primary means of communication between cells, driving cellular differentiation that develops and supports the immune system. Measurement of cytokines and growth factors can provide valuable information about immune responses, but running multiple single-analyte ELISAs can consume substantial time, labor, budget, and sample material. Moreover, ELISAs average their results across the entire sample, obscuring differential cytokine production by different subpopulations of cells.

BD T-cell cytokine kits and reagents simplify the detection of cytokines using intracellular flow cytometry on BD Accuri flow cytometers. Intracellular flow cytometry allows the rapid analysis of cytokines and other inflammatory mediators produced by multiple, phenotypically identified subpopulations within a heterogeneous sample. It can also determine whether cytokine production by an activated cell population is the result of a few cells producing large amounts of cytokine or a large cell population producing small quantities. Finally, it can measure several cytokines simultaneously for an individual cell.

The kits include or specify transport inhibitors, buffer systems, and fluorescent antibodies needed for acquisition and analysis. Free BD Accuri software templates matched to each kit include predefined workspaces, markers, regions, gates, and parameter names for quick and easy setup and analysis.

When you need to analyze a broad panel of cytokines, consider a bead-based flow cytometric immunoassay. Like ELISA assays, BD™ Cytometric Bead Array (CBA) assays measure secreted proteins produced by entire cell populations. But BD CBA can analyze 300 beads per cytokine—the equivalent of 300 ELISA wells—for up to 30 cytokines simultaneously, using very little sample. In essence, BD CBA is equivalent to running up to 30 ELISAs at once using flow cytometry.

Resources

• Product Information Sheet: BD Intracellular T-Cell Kits and Templates
• Product Information Sheet: BD™ Cytometric Bead Array (CBA) Reagents
• Webinar Replay: BD™ CBA on the BD Accuri™ C6

Sample Data

With BD Accuri flow cytometers, research laboratories no longer need a hematology analyzer to count platelets quickly and accurately.

Until 2001, platelets were commonly counted manually using a hemocytometer or automated impedance counter, despite the serious limitations of these methods. In 2001, two professional hematology organizations introduced an International Reference Method (IRM) for platelet counting based on flow cytometry. Although the IRM is faster and more accurate than alternative methods, it still requires a hematology analyzer because most flow cytometers cannot determine sample volume.

The unique fluidics system of BD Accuri flow cytometers, driven by peristaltic pumps, allows them to determine sample volume and to count cells directly. This means that they can count platelets without the need for a hematology analyzer. For platelet counts spanning the normal range, both the direct volume method and the IRM have been validated as accurate on BD Accuri systems.

Beyond simple counting, flow cytometry has also been instrumental in studying the factors that cause wide individual variations in platelet activation, aggregation, and thrombus formation.

Resources

• White Paper: Platelet Counting with the BD Accuri™ Flow Cytometer
• Webinar Slide Set: Cell and Platelet Counting Using the BD Accuri™ C6 Flow Cytometer
• Customer Interview: Paul Harrison on Platelet Counting
• Customer Interview: Christopher Jones on Platelet Dynamics

Sample Data

BD Biosciences offers an extensive selection of hundreds of antibodies to stem cell pluripotency and differentiation markers that can be combined in many ways to monitor the cells' changing expression patterns. BD Lyoplate™ screening panels can help you discover additional surface markers that characterize cells of interest.

A primary challenge of stem cell research is the inherent heterogeneity of the cell cultures. Researchers studying embryonic stem cells (ESCs) must constantly monitor the pluripotency of their cultures, while those studying human induced pluripotent stem cells (hiPSCs) must assess reprogramming success as well as pluripotency. Researchers who study, isolate, and culture mesenchymal stromal cells (MSCs) must continually monitor and verify their purity and multipotency.

Multicolor flow cytometry is ideal for this purpose because of its ability to interrogate heterogeneous cell populations, analyze their subpopulations, and characterize multiple antigens simultaneously. Each type of stem cell or derivative expresses characteristic intracellular and surface proteins that can be used for identification. Because intracellular analysis requires permeabilization, surface marker analysis is essential when researchers want to isolate live cell populations for further analysis.

Resources

• Product Information Sheet: BD Pluripotent Stem Cell Kits and Templates
• Product Information Sheet: BD Induced Pluripotent Stem Cell Kit and Template
• Product Information Sheet: BD Human Mesenchymal Stromal Cell Kit and Template
• Webinar Replay: Identification and Quality Control of Stem Cells Using Flow Cytometry(registration required)
• Customer Interview: Karina Palomares on Making Cardiomyocytes from Stem Cells

Sample Data

Immunophenotyping is one of the foremost applications of flow cytometry due to its ability to recognize different cell types based on the expression of surface and intracellular proteins. In addition to apoptosis, cell cycle, and proliferation studies, immunophenotyping is essential for understanding the cellular ecology of cancer cells.

If you don’t yet know which proteins are typically expressed by a subpopulation of interest, BD Lyoplate™ cell surface marker screening panels provide a comprehensive and efficient solution for profiling cancer cells for hundreds of human or mouse cell surface markers by flow cytometry. Deciphering the cell surface proteome enables researchers to define strategies for the analysis and isolation of targeted cells from heterogeneous populations for functional studies, drug screening, and in vivo animal studies.

Resources

• Cancer Biology Applications on the BD Accuri™ C6 Plus
• Customer Interview: Suzanne Turner on Cancer Stem Cells
• Customer Interview: Yujie Liu on Cancer Stem Cells
• Customer Interview: Emmy Verschuren on Building Cancer Models

Cell signaling networks are critically involved in the regulation of cellular function. Cell survival, growth, and differentiation are tightly regulated through expression and post-translational modification of key proteins in signaling cascades.

Many common techniques for measuring cytokine signaling and protein phosphorylation, such as Western blot and ELISA, require cell lysis, which can not only obscure the differences among individual cells in a mixed population, but can also unintentionally activate signaling pathways. Flow cytometry provides a fast, accurate, and powerful method for investigating cell signaling. Researchers can use intracellular flow cytometry to assess production of key cytokines at the single-cell level. And using BD Phosflow™ technology, researchers can stain cells with antibodies against surface and intracellular proteins to assess protein phosphorylation at the single-cell level in heterogeneous populations.

BD Phosflow standard protocol

Resources

• Product Information Sheet: BD Intracellular T-Cell Kits and Templates
• Product Information Sheet: Protein Phosphorylation Analysis Using Flow Cytometry
• Technical Bulletin: Studying Cell Signaling with the BD Accuri™ Flow Cytometer
• Webinar Replay: Enabling a Deeper Understanding of What is on, in, and Made by the Cell   PDF | Video

Changes in intracellular calcium levels regulate many important cellular functions. Because these changes can occur rapidly—in some cases within nanoseconds of stimulation—measuring them accurately is a significant research challenge. To add test compounds to the cell suspension, a “stop-flow” method is often used in which sampling is paused, the sample tube opened, the agonist added, and the tube resealed. This technique leaves a gap or blind spot in data collection that may fail to capture essential changes in Ca ²⁺ levels.

BD Accuri flow cytometers employ non-pressurized peristaltic pumps in an open fluidics system. Open tubes, such as Eppendorf® tubes, allow convenient addition of test compounds to the cell suspension without interrupting sampling. This “continuous-flow” method enables nonstop monitoring of thousands of cells and accurate dynamic Ca ²⁺ measurement of the entire population.

Resources

• White Paper: Continuous Measurement of Intracellular Calcium on the BD Accuri™ Flow Cytometer
• Webinar Slideset: Continuous Measurement of Intracellular Calcium and Other Cellular Responses

Screening thousands of cells for reporter gene expression levels is fundamental to understanding how genes are regulated inside the cell. BD Accuri systems can help you quickly examine reporter gene transfection efficiency and cell population heterogeneity—right from your benchtop. You can add additional reagents to screen cell populations, monitor DNA content, identify cells at different phases of the cell cycle, and examine heterogeneity and transient effects in reporter expression.

BD Accuri systems can measure the expression of Green, Yellow, or Red Fluorescent Protein (GFP, YFP, or RFP), as well as other fluorescent proteins, in their standard configurations. Optional filters may be used to detect signals more clearly or to resolve two fluorescent proteins with overlapping emission spectra.

Resources

• Product Information Sheet: Detecting Red Fluorescent Proteins on the BD Accuri™
• Technical Bulletin: Optical Filter Guidelines for Fluorescent Protein Analysis with the BD Accuri™ Flow Cytometer
• Technical Bulletin: Simultaneous Analysis of GFP-Transgene Expression and Plant Cell Ploidy with the BD Accuri™ Flow Cytometer

Continuous-flow addition of test compounds with the BD Accuri C6

Researchers are increasingly using flow cytometry to measure kinetic cellular processes that change over time, such as cellular response to an external event or stimulus. A new method, called continuous or “real-time” flow cytometry, involves adding an agent to a sample while simultaneously analyzing it.

The real-time method can measure almost any dynamic process and is broadly applicable in cell biology and many other fields of research. Applications include cell death, erythrocyte lysis, nanoparticle uptake, platelet activation, GFP expression, optimization of staining protocols, and disinfection of drinking water. The method has the potential to dramatically increase data richness and shed light on processes that would be difficult to assess by capturing and analyzing samples at discrete intervals.

An open, non-pressurized fluidics system makes BD Accuri systems ideal for kinetic studies. Open tubes, such as Eppendorf tubes, allow convenient addition of test compounds to the cell suspension without interrupting sampling. The real-time method enables nonstop monitoring of thousands of cells and accurate dynamic measurement of the entire population.

Resources

• Webinar Replay: Cell Biology Applications of Real-Time Flow Cytometry

Rapid and accurate quantitation of bacteria in drinking water is essential to monitor, control, and optimize treatment processes and to illuminate the biology of low-nutrient water systems. Historically, laboratories have relied on heterotrophic plate counts (HPCs) to monitor water quality, but this method is unreliable and time intensive. Flow cytometry can rapidly quantitate bacteria and discriminate them from debris.

Researchers at Eawag Aquatic Research in Zurich, Switzerland, have developed a standard flow cytometric staining protocol for BD Accuri flow cytometers to discriminate bacteria from debris in drinking water samples. Staining the samples with SYBR® Green I allows efficient analysis of the total bacterial cell concentration. Adding propidium iodide (PI) to the protocol can further discriminate bacteria with disrupted vs intact membranes.

BD Accuri systems are ideal for monitoring drinking water because of their transportability, open fluidics systems, and ability to determine sample volume and calculate cell concentrations directly. A free software template for the Eawag protocol simplifies setup and analysis.

Resources

• Product Information Sheet: BD Accuri™ Eawag Water Quality Template
• White Paper: Assessing Water Quality with the BD Accuri™ Flow Cytometer

By combining fluorescence and light scatter, flow cytometry can distinguish microbes from noise and debris. In some cases, depending on the species involved, this information might be enough to distinguish bacteria from yeast or even identify specific microorganisms in a heterogeneous sample. In other cases, additional techniques such as fluorescence in situ hybridization (FISH) can be added and even run on a flow cytometer (Flow-FISH).

Resources

• Customer Interview: Gemma Walton and Sandra Tejero on Using Flow-FISH to Identify Gut Microbes

Some authorities argue that algal biofuel is the best renewable candidate to fully replace fossil petroleum. Research laboratories racing to find or develop strains of microalgae suitable for biofuel development need efficient analytical tools to monitor algal cultures—rapidly and in real time—for size, growth, viability, chlorophyll autofluorescence, and lipid content.

Flow cytometry offers a powerful and effective method for screening microalgal cultures, and BD Accuri systems are especially well suited to the task. Microalgae can vary greatly in size, can live as single cells or in colonies, and cultures grown in open ponds often contain debris and other contaminating organisms. These characteristics can make them difficult to process through a flow cytometer’s fluidics system, even when pond water is filtered before acquisition. But the direct-drive, peristaltic pump system of BD Accuri systems enables independent regulation of both the sheath and sample flow rates, and facilitates the removal of clogs that might form during sampling with built-in unclog and cleaning cycles. Using lipophilic dyes, BD Accuri systems can rapidly estimate neutral lipid storage in small samples of live algal cells, usually without needing to remove the alga from its growth medium.

Resources

• White Paper: Multiparametric Analysis of Microalgae for Biofuels Using Flow Cytometry
• Customer Interview: Rose Ann Cattolico and James Barker on Algal Biofuel Research

Of the approximately 500,000 species of angiosperms (flowering plants) in the world, only about 2% have been described even minimally at the molecular level. Because anthropogenic change is driving many species extinct each year, a global molecular and genomic census of angiosperms is needed.

Flow cytometry can provide a rapid and efficient way of quantifying DNA content using dyes such as propidium iodide (PI). However, plant nuclear DNA content (ploidy) varies over extreme ranges, and nuclear DNA measurements of plants are often hampered by excessive cellular and subcellular debris and autofluorescence from other prevalent cellular components, such as chloroplasts.

Several factors make BD Accuri systems ideal for determining the nuclear DNA contents of plants. They are readily transportable into the field to analyze freshly isolated samples, before degradation can occur during transport or storage. The sophisticated digital signal processing system has a seven-decade dynamic range that encompasses almost the entire known span of plant C-values (haploid nuclear DNA content). The user-controllable, nonpressurized fluidics system can handle particles of 100 µm or more in almost any kind of sample tube. Finally, a blue laser optimally excites PI. A high correlation between PI-DNA emission in the FL2 and FL3 channels places the nuclear events in a narrow diagonal region from which it is easy to exclude fragments and debris.

Resources

• White Paper: Using the BD Accuri™ Flow Cytometer for Rapid and Accurate Analysis of the Nuclear DNA Contents of Flowering Plants
• Genetic Engineering & Biotechnology News Webinar Replay: Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Gene Expression
• Customer Interview: David Galbraith on Plant Genome Analysis

In industrial processes, microbes can represent contaminants to be controlled or beneficial agents to be encouraged. In the first two interviews below, researchers describe how they use BD Accuri systems to detect foodborne pathogens and monitor fermentations. In the third, a team describes how they use flow cytometry to optimize cryopreservation of semen in their efforts to preserve the endangered Asian elephant.

Resources

• Customer Interview: Byron Brehm-Stecher on Food Pathogens
• Customer Interview: Ramón Mira de Orduña on Wine Microbiology
• Customer Interview: Preserving the Endangered Asian Elephant

Many research and teaching institutions introduce graduate students to flow cytometry as part of their training and research. Increasingly, however, research professors and other academic instructors report that they are including active flow cytometry usage in their undergraduate curricula. Direct observation and experimentation are widely becoming central to a strong undergraduate education in biology as well as other disciplines.

Personal, approachable, and unintimidating, BD Accuri™ flow cytometers are ideal for both graduate and undergraduate usage. Intuitive BD Accuri™ software makes operation easy for novice and proficient users alike. The compact size and rugged design make it easy to transport into classrooms and teaching labs without optical realignment. And the low cost makes it an attractive option for individual research labs and smaller educational institutions.

Beyond coursework, undergraduates at these institutions also participate in a wide variety of research projects involving flow cytometry. Topics include lymphocyte biology, cell differentiation and development, cell culture, oxidative stress, calcium flux, metabolism, cell cycle analysis, and apoptosis and cell death. The ability to perform hands-on research using BD Accuri systems helps these young scientists understand what it's like to work in a modern research lab. And if they decide to pursue a research career, their flow cytometry experience makes them more competitive for acceptance into top-tier graduate and professional schools.

Resources

• White Paper: Sowing the Seeds of Knowledge: The Integration of Practical Flow Cytometry into Undergraduate Education
• Customer Interview: Melanie Gubbels Bupp on Education