BD Accuri News

Spotlight

Ramón Mira de Orduña on Wine Microbiology

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Ramón Mira de Orduña is professor of enology at the École d'Ingénieurs de Changins in Switzerland. His research focuses on wine analysis and microbiology, especially the nutrition and physiology of wine lactic acid bacteria and yeast. Dr. Mira de Orduña described his method of automating and controlling fed-batch fermentations, and told us what surprised him on the day he installed the BD Accuri™ C6 flow cytometer.

Read the full interview »

Application Highlight

Automating Personal Flow Cytometry

Flow cytometry's capacity to collect and process an abundance of data can also increase the effort needed to prepare, collect, and analyze samples. What can you do when a scientist's dream—a wealth of samples—seems like too much to handle?

By handling many manual steps automatically, automation can improve productivity and reduce the variability inherent in manual sample preparation. This not only allows researchers to process the proliferation of samples, but also increases data quality and reproducibility. Automation can boost throughput, making feasible screening applications such as drug discovery and gene mutagenesis. Automated flow cytometry can even enable the collection of data that would not otherwise be obtainable, such as continuous, multiparametric monitoring of cultures in a bioreactor.

A new BD Biosciences white paper explores the value of automating flow cytometry and illustrates the kinds of data an automated system can provide. The BD Accuri™ C6 personal flow cytometer offers an open communications interface that allows it to integrate smoothly with automation options from BD and other suppliers.

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Figure 1. The BD Accuri C6 with the BD CSampler accessory

The BD CSampler™ accessory (Figure 1) adds a first level of automation to the BD Accuri C6 system—simple, reliable, easy-to-use sampling with walkaway convenience. Automated sampling can significantly increase throughput, allowing researchers to run large experiments that would otherwise be infeasible. The option is compatible with both 48- and 96-well plates and 24-tube racks.

For bioprocess monitoring and other cell culture applications, BD Biosciences has worked with MSP Corporation to interconnect the BD Accuri C6 with the MSP M5000 FlowCytoPrep™ sample preparation system. The FlowCytoPrep micro-reactor platform enables user-initiated or fully automated sterile bioreactor sampling, sample preparation, and injection into the BD Accuri C6 flow cytometer. The seamless interface between the two instruments results in a turnkey, real-time, online monitoring system.

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Figure 2. Batch growth and GFP fluorescence of E. coli cells, autosampled every 15 minutes
 
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Figure 2. Batch growth and GFP fluorescence of E. coli cells, autosampled every 15 minutes
E. coli BL21 pRSET-GFP cells were grown in lysogeny broth, inoculated into a bioreactor as described as follows, and acquired and analyzed on the MSP FlowCytoPrep and BD Accuri C6. Samples were collected for 1.5 minutes at the Medium flow rate (35 μL/min), with an acquisition threshold of FSC-H = 11,000 to exclude debris. GFP fluorescence was detected in FL1 (533/30) using the standard emission filter.
A. The cultured E. coli cells were inoculated into LB in a bioreactor, grown, stirred, and analyzed every 15 minutes. Cell concentrations increased to maximum within 8 hours, while the percentage of GFP+ events decreased.
B. The cultured E. coli cells were transferred from LB to M9 minimal medium and grown overnight before similar inoculation into the bioreactor and analysis of samples every 15 minutes. Cell concentrations increased more slowly than in LB, taking nearly 30 hours to reach a maximum, while the percentage of GFP+ events increased, reached a plateau, and then decreased.

Researchers at the Biotechnology Institute of the University of Minnesota used the BD Accuri C6/FlowCytoPrep system to assess the kinetics of batch growth and GFP expression of E. coli cells cultured in either nutrient-rich lysogeny broth (LB) or M9 minimal medium. The FlowCytoPrep acquired, prepared, and delivered the samples every 15 minutes following inoculation. The BD Accuri C6 analyzed the samples for exponential growth and GFP expression as shown in Figure 2.

For drug discovery, gene mutagenesis, genotoxicity testing, and other screening applications requiring high throughput, two instruments from IntelliCyt Corporation—the HTFC® (High-Throughput Flow Cytometry) Screening System1 and the iQue™ Screener—include an integrated BD Accuri C6. Both systems offer rapid sample processing; the HTFC system, for example, can analyze a 96-well plate in less than 3 minutes or a 384-well plate in less than 12 minutes.

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Figure 3. Antibody assays on the HTFC Screening System are less variable and more reproducible
 
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Figure 3. Antibody assays on the HTFC Screening System are less variable and more reproducible
An anti-CXCR2 antibody (MAB331, R&D Systems) was tested for binding against an alanine-scan mutant panel of CXCR2 using (A) a horseradish peroxidase (HRP) immunoluminescence assay and (B) the HTFC Screening System. Data points represent the percentage of antibody binding of mutated CXCR2 normalized to wild-type protein for two replicates. Replicates that lie between the dashed lines differ less than ±40% from the linear regression, indicating a reproducible result.
Results: The HRP assay showed significant variability between replicates (r2 = 0.66), with a subset of replicates providing ambiguous data (both strong binding and no binding in one replicate pair, lying near the x-axis). In contrast, the HTFC assay showed less variability (r2 = 0.78) and no ambiguous replicate pairs, generating a highly reproducible data set and reducing the need for repeat experiments.

Integral Molecular uses the HTFC Screening System in its Shotgun Mutagenesis technology to screen libraries of hundreds to thousands of alanine scan mutations, comprising substitutions for every possible amino acid in a given protein. As Figure 3 shows, the data generated is higher quality and better correlated than results from traditional methods. The approach can be used to map epitopes and functional domains even of complex proteins such as G protein-coupled receptors (GPCRs), ion channels, and viral envelopes. The ability to map epitopes efficiently, accurately, and on structurally complex targets is an important advancement for the discovery and development of antibody therapeutics.

If your application requires even faster throughput or a higher level of automation, BD Biosciences offers a full portfolio of flow cytometry instruments and automation accessories to suit a broad range of needs. Contact your BD representative or see the instruments and sample preparation options at bdbiosciences.com.

1 As of July 1, 2013, the HTFC Screening System has been replaced by IntelliCyt's next-generation iQue Screener.

Download the white paper, Automating the Personal Flow Cytometer »


 

Tips & Tricks

Align Peaks with VirtualGain™

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Figure 4. Use VirtualGain to align peaks of different samples
 
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Figure 4. Use VirtualGain to align peaks of different samples
Chicken erythrocyte nuclei (CEN), calf thymocyte nuclei (CTN)—both from BD™ DNA QC Particles (Cat. No. 349523)— and Jurkat cells were prepared and stained with PI by standard methods. Data was collected on a BD Accuri C6 on different days. (Top row) In original data, DNA peaks from PI staining do not align. (Bottom row) VirtualGain was applied after data collection to align Peak 1 of the CEN and CTN distributions to Peak 1 of the Jurkat distribution. In column 4, overlays of DNA distributions show that after VirtualGain was applied, the peaks are aligned properly.

In certain instances, a particular fluorescence peak should have the same position across different samples or be located at a specific channel number, regardless of staining. For example, to compare DNA and cell cycle distributions of different samples stained with PI, their peaks should be aligned. With most flow cytometers, you must adjust voltage and amp gain controls to alter peak position from sample to sample.

Since BD Accuri C6 voltages are locked down at the factory, there are no gain controls that you have to adjust. Instead, you can use the VirtualGain™ tool in BD Accuri™ C6 software to align the peaks. VirtualGain mimics voltage and amp gain adjustments to grossly reposition histogram data on the x-axis after data collection.

In Figure 4, three types of cells were stained with propidium iodide (PI), but their peaks do not align sufficiently to compare their DNA distributions. In the right-hand column, VirtualGain was used to align the first peak of the CEN and CTN distributions with the first peak of the Jurkat distribution. The DNA distributions can now be compared.

VirtualGain is strictly an analysis tool and is not used while collecting data. It can be toggled on and off by clicking the asterisk under the parameter name. VirtualGain is applied only on histogram plots, and only on one parameter at a time. However, once VirtualGain is applied, you can view the transformed data in any type of plot. The tool affects only the displayed data and does not alter the raw data that is collected and saved in the FCS files.

For details on applying and using VirtualGain, see the BD Accuri™ C6 Software User Guide »

Events

BD Webinar Replay
Flow Cytometry Applications for Isolating and Analyzing Complex Heterogeneous Stem Cell Cultures »


For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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FlowCytoPrep is a trademark of MSP Corporation.

HTFC is a registered trademark, and iQue is a trademark, of IntelliCyt Corporation.