BD Accuri News

Spotlight

Byron Brehm-Stecher on Food Pathogens

Byron Brehm-Stecher

Byron Brehm-Stecher, PhD, heads the Brehm-Stecher Rapid Microbial Detection & Control Laboratory in the Department of Food Science & Human Nutrition at Iowa State University. Dr. Brehm-Stecher, who won an Accuri Creativity Award in 2010, spoke to us about developing cytometry-based assays to detect foodborne pathogens and how to get peanut butter through a flow cell.

Read the full interview »

Noteworthy

New BD Accuri™ C6 Brochure

Noteworthy - C6 Brochure

The BD Accuri™ C6 brochure has been updated with new photos, features, and data. The new version includes expanded coverage of software templates, characterization of stem cells, and water quality testing.

Download the new brochure »

Application Highlight

Multiplex Cytokine Assays with BD™ CBA

Cytokines and growth factors are a primary means of communication between cells, driving cellular differentiation that develops and supports the adaptive immune system. Different cytokines result in different cellular responses, which in turn can result in production of additional cytokines. 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.

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Figure 1. BD CBA kit analysis by analyte or sample
 
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Figure 1. BD CBA kit analysis by analyte or sample
Human peripheral blood mononuclear cells (PBMCs) were cultured for several days with plate-bound anti-CD3, soluble anti-CD27, IL-2, and IL-4. Cells were stimulated with PMA+Ionomycin for several hours before collecting culture supernatants. Samples were prepared and stained with the BD CBA Human Th1/Th2/Th17 Cytokine Kit (Cat. No. 560484), acquired on a BD Accuri C6 using the BD™ CBA Kit Accuri template, and analyzed using FCAP Array software v3.0.1. A. Bar chart of human IFN-γ levels for all standards and test samples. B. Bar chart of all seven cytokine levels for one sample.

Bead-based flow cytometric immunoassays are powerful methods of quantifying cytokines because they allow researchers to multiplex many analytes with very little sample. A BD™ Cytometric Bead Array (CBA) assay contains a cocktail of beads bound with specific capture antibodies that differ slightly in fluorescence intensity. The beads are mixed with samples along with PE-labeled detection antibodies and run on a BD Accuri™ C6 flow cytometer. BD CBA can analyze 300 beads per cytokine—the equivalent of 300 ELISA wells—for up to 30 cytokines simultaneously. In essence, BD CBA is like running multiple ELISAs at once using flow cytometry.

There are two ways to use BD CBA: kits and flex sets. BD CBA kits provide preconfigured panels of 3–7 analytes for ultimate ease of use. For example, the BD™ CBA Human Th1/Th2/Th7 Cytokine Kit can quantitate IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A simultaneously. Other kits include panels of human cytokines, chemokines, and anaphylatoxins; mouse cytokines and immunoglobulins; and non-human primate cytokines.

On the BD Accuri C6, the beads are excited by the red laser and detected in FL4, while the PE reporter is excited by the blue laser and detected in FL2. A BD Accuri™ C6 software template simplifies setup and acquisition. As Figure 1 shows, results can be displayed in FCAP Array™ software by bead (graphing one analyte across all samples) or by sample (graphing one sample across all analytes).

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Figure 2. BD CBA flex set analysis on the BD Accuri C6
 
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Figure 2. BD CBA flex set analysis on the BD Accuri C6
Human PBMCs were cultured for several days with plate-bound anti-CD3, soluble anti-CD27, IL-2, and IL-4. In one experimental condition, cells were stimulated with PMA+Ionomycin for several hours before harvesting. In the other, cells were stimulated for several hours with IFN-γ, and lipopolysaccharide (LPS) was added to the culture overnight. All samples were prepared using the BD™ CBA Human Soluble Protein Master Buffer Kit (Cat. No. 558264) and stained with flex set reagents for 10 different cytokines. Samples were acquired on a BD Accuri C6 in the 2-blue, 2-red configuration using the BD CBA Flex Set Accuri template and analyzed using FCAP Array software v3.0.1. A. Bar chart of human IL-2 levels for all standards and test samples. B. Comparison of all 10 cytokine levels for one sample under both experimental conditions.

BD CBA flex sets enable analysis of up to 30 analytes. Available specificities include soluble protein assays for detection of human, mouse, or rat cytokines, chemokines, and growth factors; human immunoglobulins; cell signaling assays; and enhanced sensitivity assays that can detect protein concentrations of 1.0 pg/mL or less. The beads contain two dyes excited by the red laser that create 30 unique bead populations, while a PE reporter quantifies the analytes. Flex sets are completely configurable, and the workflow essentially allows researchers to build their own multiplex kit.

Flex sets require the optional Selectable Lasers Module to reconfigure the BD Accuri C6 so that two detectors (instead of the usual one) detect signal from the red laser. Again, a software template simplifies setup and acquisition, and data analysis is performed in FCAP Array software. Figure 2 illustrates how you might compare production of 10 different cytokines by human blood cells in response to stimulation with PMA+Ionomycin vs IFN-γ.

Replay the webinar, BD™ CBA on the BD Accuri™ C6: Bringing Multiplexed Detection to the Benchtop »


 

Tips & Tricks

Use BD FACSelect™ to Design Multicolor Panels

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Figure 3. Search for antibodies in BD FACSelect
 
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Figure 3. Search for antibodies in BD FACSelect
To design a panel with the BD FACSelect tool, select your sample species, target specificities, and the lasers or fluorochromes you want to use. For the BD Accuri C6, select the boxes for Blue and Red lasers.

It’s not always easy to select the best reagents for a multicolor experiment from BD’s broad portfolio. The BD FACSelect™ Multicolor Panel Designer can help.

You’ll find the BD FACSelect tool on BD’s Multicolor Flow Cytometry Tools page. Using the Designer is as simple as completing two short forms. The first (Figure 3) lets you select your reactivity (sample species), target specificities (markers) or clones, and available lasers. For the BD Accuri C6, select the blue and red lasers and click Search.

For each laser, you’ll see a table (Figure 4) that shows the number of available conjugates for each fluorochrome/marker combination. Click the arrow to see the clones or other options, and select one to add it to your panel on the right. You can bookmark or print the panel, or add the reagents to your shopping cart.

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Figure 4. Build your panel in BD FACSelect
 
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Figure 4. Build your panel in BD FACSelect
Each number represents the conjugates available for that marker in that fluorochrome. Click the arrow to see the options and select one to add it to your panel. You can bookmark or print the panel, or add the reagents to your cart. Note that certain fluorochromes, such as APC-H7 and APC-Cy™7, may work better with optional filters; consult the BD Accuri™ C6 Flow Cytometer Optical Filter Guide for advice.

You can use multicolor design principles to pair markers with fluorochromes to help your experiment clearly distinguish populations of interest. For discussions of design principles on the BD Accuri C6, consult the links at the end of this article.

You’ll find other useful design resources on the Tools page as well, including our Fluorescence Spectrum Viewer, Example Human and Mouse Panels, and the BD Antibodies iPad® App.

Read more about multicolor panel design on the BD Accuri C6 »

Replay the webinar, Multicolor Flow Cytometry - Setup and Optimization on the BD Accuri™ C6 Flow Cytometer »


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Alexa Fluor® is a registered trademark of Life Technologies Corporation.

Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.

FCAP Array is a trademark of Soft Flow Hungary Ltd.