BD Accuri™ C6 Plus

Introducing the BD Accuri C6 Plus

The BD Accuri C6 Plus personal flow cytometer is the newest generation of the BD Accuri platform. Enhanced sensitivity, reliability, and capabilities bring flow cytometry even more within reach for new and experienced flow cytometry researchers.

With its compact 11 x 14.75 x 16.5-inch footprint, light weight of 30 lb, and operational simplicity, the BD Accuri C6 Plus supports a wide array of applications including immunology, cell and cancer biology, plant and microbiology, and industrial applications.

BD Accuri C6 - Information and resources for the predecessor BD Accuri C6 personal flow cytometer

Features

Enhanced sensitivity, reliability and capability

  • Highly sensitive: MESF <75 (FITC) and <50 (PE)
  • Reliable instrument performance with automated QC
  • Optional laser filters for expanded capability
  • Volumetric counting and continuous sampling

Simplicity: easy to learn, use, maintain and teach

  • Quick and easy instrument setup
  • Intuitive software with automated functions
  • Simplified workflow with BD quality reagents and kits
  • Walkaway convenience with automated sample loader

Compact and transportable

  • Small footprint for easy transportation between labs
  • Allows researcher to go places and explore

Wide applications in areas such as immunology, cell and cancer biology, plant and microbiology, and education

  • Cell cycle, apoptosis, proliferation, viability, signaling, DNA damage, DNA content
  • BD Accuri™ flow cytometers have been cited in more than 7,500 Google Scholar publications*

* Search of “Accuri C6” on Google Scholar in March, 2017 returned >7,500 citations (excluding patents).

The BD Accuri CSampler Plus automation accessory

For walkaway convenience, the optional BD CSampler Plus accessory offers reliable and easy-to-use automation. The system supports 48- and 96-well plates and deep-well plates, and is also supplied with a 24-tube rack for standard 12 x 75-mm tubes. They are processed directly in the BD Accuri C6 Plus cytometer, saving time. The BD CSampler Plus adds a minimal footprint to the BD Accuri C6 Plus, about three feet square for the pair, keeping your benchtop free for other uses.

Simplicity and Sensitivity on your Benchtop

The BD Accuri™ C6 Plus is equipped with a blue and red laser, two light scatter detectors, and four fluorescence detectors with optical filters optimized for the detection of many popular fluorochromes, including FITC, PE, PerCP-Cy™5.5, and APC, as well as newer polymer dyes such as BD Horizon Brilliant™ Blue 515. The BD Accuri C6 Plus can also analyze many variants of fluorescent proteins, such as GFP, YFP, and mCherry. Its adaptability and suitability for experiments up to four colors have allowed researchers to publish numerous scientific articles citing BD Accuri flow cytometers.

During manufacture, laser and optical alignments are set and locked down. With no need to adjust detector voltage, the system is easier to use. Data is digitally collected over a wide dynamic range and is fully available to users as needed, eliminating the risk of data loss due to incorrect settings. Software functions such as Zoom and VirtualGain™ allow visualization of data at any scale, so that users can precisely set gates and regions.

Despite its compact size, the optical system demonstrates extraordinary fluorescence sensitivity. Intensive testing ensures that the optical and fluidic design can withstand rugged conditions. Provided the system is anchored, it can run samples even if the benchtop is in motion, such as aboard a ship.

BD Accuri C6 Plus optical bench

A compact optical design, fixed alignment, and pre-optimized detector settings make the system easier to use.

Filter, laser, and configuration options provide added flexibility

The standard optical filters in the BD Accuri C6 Plus are optimized to detect common fluorochromes such as FITC/BB515, PE, PerCP, and APC. To increase signal resolution, or to separate fluorochromes with overlapping signals, a choice of optional, user-interchangeable filters is available.

BD Accuri C6 Plus fluorochrome support

All fluorochromes in the table can be detected by the BD Accuri C6 Plus equipped with standard filters. Optional filters can be used to increase resolution or to separate fluorochromes with overlapping signals, like GFP and YFP.

In the standard configuration of the BD Accuri C6 Plus, three detectors read fluorescence emissions from fluorochromes excited by the blue laser, while the fourth detector reads emissions from fluorochromes excited by the red laser (3-blue/1-red). By installing the optional Selectable Laser Module, you can operate the system in 2-blue/2-red and 4-blue configurations. This option significantly expands the fluorochrome combinations you can analyze.

Selectable Laser alternate configurations

The optional Selectable Laser Module allows two or four fluorescence channels to be assigned to the blue laser, vs three in the standard configuration. The remaining channels (if any) are assigned to the red laser.


Four-color whole blood immunophenotyping panel on the BD Accuri C6 Plus

A unique, non-pressurized, peristaltic pump system drives the fluidics. The system accurately monitors the sample volume pulled per run, and can calculate absolute counts or sample concentration per µL without the use of counting beads. These absolute counts are more precise and far less tedious than manual counts.

The system supports a wide array of sample tubes. Fluidics are cleaned automatically on instrument shutdown, and laboratory-grade water is used as sheath fluid, reducing operating costs.

For walkaway convenience, the optional BD CSampler™ Plus accessory offers reliable and easy-to-use automation. The BD CSampler Plus adds minimal footprint to the BD Accuri™ C6 Plus.

Sample Flexibility with Optional Walkaway Sample Loading

BD CSampler Plus automatic sampling accessory

The optional accessory offers reliable, easy-to-use automation while adding minimal footprint. The BD CSampler Plus supports:

  • 48-well plates
  • 96-well plates
  • 96-deep-well plates
  • 24-tube rack (included) for standard 12 x 75-mm tubes

Faster, simpler protocol with continuous sampling capability

Continuous sampling on the BD Accuri C6 Plus


Bacterial viability on the BD Accuri C6 Plus

Bead-free cell counts in the lab and in the field

The unique fluidics system of the BD Accuri C6 Plus, driven by peristaltic pumps, allows it to determine sample volume and count cells rapidly, directly, and automatically from the software, eliminating laborious plate counts. Direct counts on the BD Accuri C6 Plus correlate highly with counting beads and are more precise than manual counts.

Intuitive Software—Master in Minutes

BD Accuri™ C6 Plus software features an intuitive user interface. Even novice flow cytometry users find it so easy to learn and use that most can collect and analyze data in less than an hour. Software options and instrument controls are clearly visible from the software’s tabbed interface.


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.

Accuri C6 Plus - Analyze
Accuri C6 Plus - Statistics
Accuri C6 Plus - Batch Analysis

Automated instrument QC

Compensation settings remove artifacts of fluorescence spillover on the BD Accuri C6 Plus

Instrument QC Compensation

After each time Instrument QC is performed, the software also updates compensation settings for FITC, PE, APC, and PerCP or PerCP-Cy™5.5. Instrument QC Compensation Settings can be selected from the Compensation Preferences window as an easy and convenient starting point for proper compensation controls.

Alternatively, you can select User Defined Compensation Settings to calculate compensation manually, which is also necessary for other fluorochromes or dyes.

As Easy as Cell Analysis Is Going to Get

Now it’s even easier to apply the power of flow cytometry to your research with ready-to-go reagent kits and free software templates specific to your studies.

Find out how this powerful platform, combined with BD kits and templates, will give you more insight and easier workflow.

Preconfigured kits—for immunophenotyping, apoptosis, cell cycle, microbial counting, intracellular cytokine studies, stem cell analysis, and more—include fluorescent antibodies, buffer systems, and other reagents needed for acquisition and analysis. BD Accuri™ software templates matched to each kit include predefined workspaces, markers, regions, gates, and parameter names for quick and easy setup and analysis.


All reagents and kits are compatible with both BD Accuri™ C6 Plus and Accuri™ C6 flow cytometer systems. Select from the following table:

BD Accuri C6 system (32-bit) software is not forward compatible with BD Accuri C6 Plus (64-bit) software and the FCS 3.1 file format.

Category Product Information Sheet Brand Kit Cat. No. C6 Plus Template
Cancer and Cell Biology BD Apoptosis Kits and Templates BD Pharmingen™ Annexin V FITC Apoptosis Detection Kit II 556570 Download
BD Pharmingen™ Annexin V PE Apoptosis Detection Kit I 559763 Download
BD™ MitoScreen (JC-1) Kit 551302 Download
BD Pharmingen™ Caspase-3 PE Assay Kit 550914 Download
BD Pharmingen™ Caspase-3 FITC Assay Kit 550480 Download
BD Apoptosis, DNA Damage and Cell Proliferation Kit and Template BD Pharmingen™ Apoptosis, DNA Damage and Cell Proliferation Kit 562253 Download
BD Cell Cycle and DNA Kits and Templates
BD Cycletest™ Plus DNA Reagent Kit 340242 Download
BD Pharmingen™ FITC BrdU Flow Kit 559619 Download
BD Pharmingen™ APC BrdU Flow Kit 552598 Download
Category Product Information Sheet Brand Kit Cat. No. C6 Plus Template
Immunology BD Naïve/Memory T-Cell Kits and Templates BD Multitest™ CD45RA/CD45RO/CD3/CD4 340571 Download
BD Multitest™ CD45RA/CD62L/CD3/CD4 340977 Download
BD Pharmingen™ Human Naïve/Memory T Cell Panel 561438 Download
BD Intracellular T-Cell Kits and Templates
BD Pharmingen™ Th1/Th2/Th17 Phenotyping Kit 560751 Download
BD FastImmune™ IFN-γ/CD69/CD8/CD3 Kit 346048 Download
BD FastImmune™ IFN-γ/IL-4 Kit 340456 Download
BD Regulatory T-Cell Kits and Templates
BD Pharmingen™ Human Regulatory T Cell Cocktail 560249 Download
BD Pharmingen™ Human Th17/Treg Phenotyping Kit 560762 Download
BD Pharmingen™ FoxP3 Staining Kit 560132 Download
BD Mouse B-Lymphocyte Kits and Templates
BD Pharmingen™ Mouse B-Lymphocyte Subset Antibody Cocktail 558332 Download
BD Pharmingen™ Mouse B-Lymphocyte Activation Antibody Cocktail 558063 Download
BD Mouse T-Cell Cytokine and Treg Kits and Templates
BD Pharmingen™ Mouse Th1/Th2/Th17 Phenotyping Kit 560758 Download
BD Pharmingen™ Mouse Th17/Treg Phenotyping Kit 560767 Download
BD Mouse T-Lymphocyte Kits and Templates
BD Pharmingen™ Mouse T-Lymphocyte Subset Antibody Cocktail 558431 Download
BD Pharmingen™ Mouse T-Lymphocyte Activation Antibody Cocktail 557908 Download
Category Product Information Sheet Brand Kit Cat. No. C6 Plus Template
Stem Cells BD Pluripotent Stem Cell Kits and Templates BD Stemflow™ Human Pluripotent Stem Cell Sorting and Analysis Kit 560461 Download
BD Stemflow™ Human Pluripotent Stem Cell Transcription Factor Analysis Kit 560589 Download
BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit 560477 Download
BD Induced Pluripotent Stem Cell Kit and Template BD Stemflow™ Human iPSC Sorting and Analysis Kit 562626 Download
BD Human Mesenchymal Stromal Cell Kit and Template BD Stemflow™ Human MSC Analysis Kit 562245 Download
Category Product Information Sheet Brand Kit Cat. No. C6 Plus Template
Bead-Based Assays BD™ Cytometric Bead Array (CBA) Reagents BD™ CBA Kits See Kits Download
BD™ CBA Flex Sets See Flex Sets Download
Category Product Information Sheet Brand Kit Cat. No. C6 Plus Template
Microbiology BD Accuri™ Eawag Water Quality Template Download

Once you have downloaded a template, select File > Open Workspace or Template and open it. Gate positions, zoom level, thresholds, and compensation, run, and fluidics settings are all fully adjustable and may require optimization for different sample types. Once the settings are optimized for your experiment, click Run to begin data acquisition.

Note: The linked templates on this page are provided for the convenience of our research customers. All reagents and kits are compatible with both BD Accuri C6 Plus and BD Accuri C6 systems. The product information sheets and the catalog of available kits and templates, and data within them, were generated using the BD Accuri C6 flow cytometer system. Further optimization may be required before use, and it is the end user's responsibility to ensure that use of the template is appropriate to his or her research.

Overview

Among the array of applications that facilitate immunology and stem cell studies, multicolor flow cytometry is preeminent. Immunophenotyping was one of the first applications of flow cytometry, and for over 20 years, BD Biosciences has actively supported groundbreaking research in the field with flow cytometry systems and reagents.

Using panels of directly conjugated fluorescent antibodies to recognize specific cell surface and intracellular epitopes, multicolor flow cytometric analysis allows researchers to interrogate specific target protein levels expressed by individual cells in various phases of development and differentiation. BD's solutions for cell identification, transcription factor expression, and cytokine secretion and measurement reflect a commitment to high quality and consistency needed for advanced research.

BD Accuri™ personal flow cytometers bring immunology and stem cell studies to the benchtop. With two lasers, two scatter detectors, and four fluorescence detectors, they can handle most common assays with ease. Two additional features further streamline the process:

Absolute counts. Cell concentration can be calculated directly from BD Accuri™ software statistics tables without the addition of counting beads.

Kits and templates. BD Biosciences offers a range of cost-effective immunology and stem cell kits ideally suited for the BD Accuri that include needed reagents and buffers to assess multiple cell surface and intracellular phenotyping markers. Free BD Accuri software templates simplify data collection and analysis.

The following sections and resources illustrate the rich data you can generate by using BD Accuri systems for immunology and stem cell applications.

Resources

 

Immunophenotyping

Flow cytometry can rapidly and accurately immunophenotype many kinds of cells, including lymphoid tissue (thymus, spleen, and lymph nodes), digested solid tissues, and blood.

For example, one common task in human blood analysis is to identify and enumerate populations of platelets, lymphocytes, monocytes, granulocytes, and eosinophils. Flow cytometry is a quick and reliable methodology for identifying these populations simultaneously in a single sample, based on their immunophenotypes (see table).

Several features of BD Accuri flow cytometers make them ideal platforms for differential analysis of human peripheral blood samples. First, fixed-voltage detectors simplify data collection and reduce the potential for data loss due to signal over- or under-amplification. Second, the instruments' broad dynamic range makes it easy to analyze populations as varied in size as platelets and eosinophils in the same data file. Third, the easy-to-use software Zoom tool allows precise control when setting gates. Finally, direct volume measurement allows direct calculation of the cell concentration per µL for each population of interest.

Population Immunophenotype
Platelets CD41 +CD45 -
Lymphocytes CD3 +CD45 +
Monocytes CD11b +CD14 +
Granulocytes CD11b +CD14 -
Eosinophils FL1 ++SSC high

Resources

Sample Data

T-cell phenotyping data analyzed on a BD Accuri C6

Identification and gating of five peripheral blood cell populations

Using the Zoom tool to adjust gates

Differentiating naïve and memory T cells using the kit and software template

Distinguishing Tregs with CD127 using the Treg cocktail and template

Identifying three lymphocyte populations using two detectors

Cell Counting

 

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

Sample Data

Absolute cell counts measured by direct volume vs counting beads

Cytokine Analysis & Bead-Based Immunoassays

 

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

Sample Data

Assessing Th1, Th2, and Th17 cytokines using the phenotyping kit and template

Assessing cytotoxic T-cell cytokines using the kit and template

Cytokine analysis by analyte or sample using BD CBA kits

Identification and gating of five peripheral blood cell populations

Platelets

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

Sample Data

Validation of platelet counting methods on the BD Accuri C6

Stem Cells

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

Sample Data


Stem cell analysis using the pluripotent stem cell kit and software template

Assessing iPSC pluripotency and reprogramming using the iPSC kit and template

Verifying the MSC phenotype using the hMSC kit and template

Overview

Cellular processes and events are probabilistic and take place in a context of complex varying environments, which they influence and which influence them. For this reason, flow cytometry is an ideal methodology for cell and cancer biology research since it supports the multiparametric analysis of individual cells and subpopulations in heterogeneous samples.

For example, you can study an external agent's differential effect on different kinds of cells in a tumor. You can analyze cell cycle and aneuploidy or trace signaling cascades across multiple subpopulations. Results are rapid and resolve data at the single-cell level rather than averaging across a sample.

A personal flow cytometer in the lab provides many advantages for cell and cancer biology studies. When cells are ready for analysis or rare tumor samples arrive, it's crucial to have a flow cytometer at hand, ready to go. With two lasers and four fluorescence detectors, BD Accuri personal flow cytometers bring this versatility and convenience to your benchtop. Two additional features can further simplify your research:

Absolute counts. Cell concentration can be calculated directly from BD Accuri™ software statistics tables without the addition of counting beads.

Kits and templates. BD Biosciences offers a range of cost-effective cell biology kits ideally suited for the BD Accuri that include needed reagents and buffers to assess apoptosis, cell cycle and proliferation, and DNA damage. Free BD Accuri software templates simplify data collection and analysis.

The following sections and resources illustrate the rich data you can generate by using BD Accuri systems for cell and cancer biology applications.

Resources

Apoptosis, Cell Cycle, DNA Damage, and Proliferation

Many factors, including stress, radiation, environmental exposure, and treatment with small molecules, can lead to changes in cell cycle, apoptosis, DNA damage, and cell proliferation. Detecting these events has broad application in the fields of cell biology, cancer research, and developmental biology.

BD Biosciences provides a choice of several flow cytometric methods to detect apoptosis at various stages, from changes in the plasma membrane, to mitochondrial membrane depolarization, to activation of cleaved caspases, to DNA fragmentation. Apoptotic cells can be detected alone or simultaneously along with DNA damage and cell proliferation.

Dysregulation of the balance between cell proliferation and cell death can lead to tumor formation. Therefore, understanding the molecular signals that regulate cell cycle and apoptosis is key to the advancement of cancer research. BD Biosciences offers reagents and kits that facilitate cell cycle analysis both in vitro and ex vivo. These DNA intercalating agents and nucleotide analogs allow researchers to measure cellular DNA content, separate cells into G 0/G 1, S, and G 2/M phases, analyze DNA ploidy, and detect DNA synthesis in actively dividing cells.

Resources

Sample Data

Apoptosis detection on the BD Accuri C6 Plus

Cell cycle analysis using BrdU flow kits and software templates

Cell cycle and ploidy analysis on the BD Accuri C6

Detecting mitochondrial depolarization using BD Pharmingen MitoStatus reagents

Detecting apoptosis, DNA damage, and cell proliferation using the kit and template

Cancer Research

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

Sample Data

Immunophenotyping breast cancer cell lines for cancer stem cell markers

Cell Signaling

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.

Cell Signaling - Phosflow Protocol Graphic
BD Phosflow standard protocol

Resources

Cell signaling analysis using BD Phosflow reagents

Calcium Flux

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

Continuous sampling on the BD Accuri C6 Plus

Gene Expression

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

Simultaneous detection of multiple fluorescent proteins

Detection of green and yellow fluorescent proteins

Kinetics

Kinetics - Continuous Flow Graphic
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

Continuous, gap-free recording of intracellular calcium levels on the BD Accuri C6

Interaction of nanoparticles with mammalian cells

Continuous sampling at varying temperatures

Overview

In the diverse field of microbiology research, flow cytometry is a powerful technique for analyzing microorganisms including bacteria and yeast, and offers many advantages over more conventional techniques. Light scatter data can reveal basic information about microbes' size, shape, and surface features, while fluorescent stains can assess microbes' cell viability, metabolic activity, and concentration. In some cases, this information might be enough to identify specific microorganisms in a heterogeneous sample. In others, additional techniques such as fluorescence in situ hybridization (FISH) can be added.

These characteristics allow flow cytometry to be used in diverse microbiological applications, such as measurement of gene expression, monitoring bacterial and yeast fermentations, recombinant protein production in bacteria, and food processing. On BD Accuri™ flow cytometers, triggers and thresholds help to accurately identify small particles such as microbes. Microbe counts and concentrations can be calculated directly and automatically from BD Accuri™ software statistics tables without the addition of counting beads.

The following sections and resources illustrate the rich data you can generate by using BD Accuri systems for microbiology applications. See also the separate tab for Industrial Applications that use microbes for bioprocessing, food and beverage processing, and biofuels.

Resources


Microbe Counts and Viability

Perhaps the most common task in microbial analysis is to identify and count microbes. On BD Accuri flow cytometers, triggers and thresholds help you accurately identify small particles such as microbes and distinguish them from instrument noise, background signals, and debris. Microbe counts and concentrations can be calculated rapidly, directly, and automatically from the software, eliminating laborious plate counts. If microbes are transfected with green, yellow, red, or other fluorescent proteins, they can be detected and clearly differentiated from non-transfected populations.

The BD™ Cell Viability Kit provides a simple, two-color method to monitor microbial cell viability on BD Accuri flow cytometers. Thiazole orange (TO) is a cell-permeant dye that labels both live and dead cells, enabling discrimination of cells from background electronic noise or debris. Propidium iodide (PI) is impermeable to healthy cells with intact membranes, but permeates cells with compromised membranes such as dead cells. When used in combination in the kit, these dyes provide a rapid, simple method to distinguish live, dead, and injured bacteria, yeast, or eukaryotic cells.

Resources

Sample Data

Bacterial viability on the BD Accuri C6 Plus

Resolution of four microbial populations using light scatter and an autofluorescence trigger

Live/dead discrimination of E. coli using the BD™ Cell Viability Kit

Detection of GFP expression in bacteria

Water Quality Analysis

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

Algae analysis

Water quality analysis using the Eawag water quality template

Water samples analyzed on the BD Accuri C6

Aquatic Research

Environmental research on marine and freshwater ecosystems often focuses on their microbiomes. The productivity of phytoplankton and cyanobacteria species responsible for harmful algal blooms is of critical concern. Fortunately for researchers, most aquatic microorganisms contain natural chlorophylls, phycobilins, and other intrinsic fluorescent pigments (see table) that can readily be detected by flow cytometry.
Fluorophore Exciting Laser Major Emission Wavelength C6 Detector (filter)
Chloropyll a,b 488 640 nm FL3 (670 LP)
Phycoerythrin 488 575 nm FL2 (585 ±20)
C-phycocyanin 640 650 nm FL4 (675 ±12.5)
R-phycocyanin 640 646 nm FL4 (675 ±12.5)
Allophycocyanin 640 660 nm FL4 (675 ±12.5)

Cyanobacterial profiling: Lake Michigan

Autofluorescence profiles for three phytoplankton species on the BD Accuri C6

Phytoplankton counts in oyster nursery inflows and outflows

Microbial Species Identification

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

After fluorescence gating, light scatter can distinguish bacteria and yeast

Determination of bacterial strain by PNA FISH

Overview

Many modern industries employ biological processes that require precise monitoring and control. Bacterial bioprocessing produces ingredients for food, drugs, cosmetics, and more; algae can be used to manufacture biofuels; yeast fermentation produces wine and beer. By offering rapid analysis of heterogeneous samples, flow cytometry is an ideal method for monitoring these processes.

BD Accuri™ flow cytometers offer particular advantages for industrial and agricultural applications. Compact and rugged, they can be transported into labs and processing facilities, and even into the field. The open fluidics system allows it to connect easily to bioreactors and fermentation units for automatic sampling and continuous culture monitoring.

The following sections and resources illustrate the rich data you can generate by using BD Accuri systems for industrial applications in bioprocessing, biofuels, agriculture, fermentation, and other enterprises.


Bioprocessing

Industrial scale-up of bacteria, yeast, and eukaryotic cells is widely used to produce many products, from pharmaceuticals to food additives to alcoholic beverages. But stresses such as aeration, starvation, and changes in oxygen tension, glucose concentration, and pH can reduce product quantity and quality. Precise control and optimization of cell growth are crucial.

Flow cytometry offers a powerful and effective methodology for monitoring cellular status and growth in bioreactors by rapidly characterizing complex cultures at the single-cell level. Flow cytometry applications common in bioprocessing include cell counting, viability, cell cycle, apoptosis, transfection efficiency, cell line development, and media optimization.

Offering both performance and simplicity, BD Accuri systems allow bioprocess engineers to use light scatter signals to discriminate cells and fluorescence signals to measure cell viability, vitality, and other important cell characteristics. BD Accuri systems also interface smoothly with automated bioreactor sampling systems so that samples can be taken continuously and analyzed automatically. Bioprocess engineers can use the resulting real-time, online bioreactor data for pinpoint process control.

Resources

Sample Data

Using apoptotic cell counts to trigger passage of a CHO cell culture

Batch growth and GFP fluorescence of E. coli cells, autosampled every 15 minutes

Biofuels

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

Light-scatter profiles for microalgae of varying size

Neutral lipid content in Isochrysis sp., analyzed with Nile Red

Plant Biology

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

Sample Data

Distinguishing DNA content and ploidy in A. thaliana root tissues

Food, Beverage, and Veterinary

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

Education

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

Sample Data

Education - Student Photo

Flow cytometry immunophenotyping lab in an undergraduate immunology course

An undergraduate experiment on compound research effects at CSU Northridge

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  • Flow Cytometry Application in CRISPR-Cas Systems and Molecular Biology Assays   PDF | Video
  • Introducing the New BD Accuri™ C6 Plus System: A Powerful and Versatile Personal Flow Cytometer Tool for Life Science Research   PDF | Video

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All reagents and kits are compatible with both the BD Accuri C6 Plus and BD Accuri C6 flow cytometer systems. Platforms referred to as "BD Accuri" represent both the BD Accuri C6 Plus and BD Accuri C6. Data was generated on either the BD Accuri C6 Plus or the BD Accuri C6 as indicated in figure legends.


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All reagents and kits are compatible with both the BD Accuri C6 Plus and BD Accuri C6 flow cytometer systems. Platforms referred to as "BD Accuri" represent both the BD Accuri C6 Plus and BD Accuri C6. Data was generated on either the BD Accuri C6 Plus or the BD Accuri C6 as indicated in figure legends.

BD Accuri C6 system (32-bit) software is not forward compatible with BD Accuri C6 Plus (64-bit) software and the FCS 3.1 file format.

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