BD Accuri News

 

March 2015


Spotlight

Dorothee Bienzle on Diagnosing Veterinary Lymphomas

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Dorothee Bienzle is professor of pathobiology in the Ontario Veterinary College at the University of Guelph, ON. Her research interests focus on the pathogenesis of retroviral infection, mechanisms of inflammation, and development of new diagnostic assays. Dr. Bienzle told us how her research on flow cytometric analysis of lymph node aspirates evolved into a diagnostic veterinary assay used around the world, which she now runs on her lab’s new BD Accuri™ C6 flow cytometer.

Read the interview »

Noteworthy

Join Us at AACR in Philadelphia

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Join BD April 19–22 in Philadelphia, PA, at the 2015 meeting of the American Association for Cancer Research. Visit Booth #745 during exhibition hours to test drive a BD Accuri C6, or drop by between 4 PM and 5 PM on April 20 or 21 to meet senior BD scientists and discuss your research needs. Three teams of BD researchers will also be presenting papers at the meeting.

Application Highlight

Integrating Flow Cytometry Into Education

Can undergraduates learn flow cytometry? Ten years ago, the question would have seemed ludicrous. The technology was too complicated and the equipment too expensive to entrust to undergraduates.

But flow cytometry is a technology of growing importance in biology and many related fields. Integrating flow cytometry into undergraduate and graduate curricula is essential for preparing students to work in research labs or to conduct their own independent research. And now, with the introduction of personal flow cytometers such as the BD Accuri C6, this key technology is finding its way into teaching labs and classrooms at the graduate and even undergraduate level.

At a recent BD Biosciences webinar, two leading educators described successful methods of integrating flow cytometry into education. In their view, the BD Accuri C6 can help students concentrate on learning the concepts and value of flow cytometry without worrying about the technology.

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Victoria Robinson, an undergraduate at Randolph-Macon College, acquires samples on a BD Accuri C6

Dr. Melanie Gubbels Bupp, an immunologist and assistant professor of biology at Randolph-Macon College in Ashland, VA, described how she incorporates flow cytometry into her courses for undergraduate biology majors. In one four-week lab, the students inject mice with 6% thioglycollate in PBS (or PBS control only) to induce peritoneal inflammation, so that they can determine the order in which different leucocyte subsets are recruited to the site. They then acquire the cells on the BD Accuri C6, and Dr. Gubbels Bupp explains—using some ingenious animations that you can see at the 22-minute mark in the webinar replay—how flow cytometry works and how to read a dot plot. Dr. Gubbels Bupp recommended holding off on explaining the technology until the moment students need to know it. Front-loading technical information beforehand can be overwhelming, she maintained, and often ineffective.

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Figure 1. Two student teams attempt to manage E. coli growth by controlling the glucose feed rate
 
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Figure 1. Two student teams attempt to manage E. coli growth by controlling the glucose feed rate
Two teams of MSc students at the University of Birmingham, UK, were assigned a practicum in which they attempted to grow E. coli cells in 150-L fed-batch bioreactors over the course of a week. Students were encouraged to develop their own analysis methods and strategies, including flow cytometry on the BD Accuri C6. The plots show analysis of the cultures over time using PI and BOX to assess viability and membrane depolarization, respectively. Team 2 (lower plots) was able to control the glucose feeding rate to maintain a large percentage of healthy cells. Team 1’s feeding regime produced more unhealthy and dead cells. Data courtesy of Tim Overton, School of Chemical Engineering, University of Birmingham, UK.

Dr. Tim Overton, a lecturer at the University of Birmingham, UK, who won a BD Accuri Creativity Award for teaching in 2011, described how he uses the BD Accuri C6 to teach fermentation and cell culture for bioprocessing to masters-level students in biochemical engineering. In one practical, teams of students try to manage the growth of E. coli in 150-L fed-batch bioreactors over the course of a week. They employ flow cytometry to assess bacterial physiology using the fluorescent dyes propidium iodide (PI) and DiBAC4(3) (BOX) to measure viability and membrane depolarization, respectively. In the results shown in Figure 1, Team 2 was able to control the glucose feed rate so that most growing, multiplying cells remained healthy. Team 1 was not so successful; they fed the cells too much sugar, which the cells turned into acid, which in turn killed some of the cells.

Once trained in flow cytometry, students at both institutions have continued to use the BD Accuri C6 in independent research projects. Last November, three Randolph-Macon undergraduates presented papers on cellular immune responses to malnutrition at the Autumn Immunology Conference (AIC) in Chicago, IL.

Both educators agreed that, although their students come from diverse backgrounds and learn at different rates, they are very interested in learning how to use flow cytometry. In many cases, students step up and teach each other what they may not know about optics or fluorescence. Students find the BD Accuri C6 to be a friendly, approachable, intuitive instrument that turns out to be ideal for learning about flow cytometry.

Replay the webinar »

Read interviews with Melanie Gubbels Bupp and Tim Overton »

Download the white paper, Sowing the Seeds of Knowledge: The Integration of Practical Flow Cytometry into Undergraduate Education »


 

Tips & Tricks

Excluding Dead Cells From Analysis

When counting cells, and in many other applications, it is often important to exclude dead cells from your analysis. For example, suppose you are conducting a cancer study by injecting live, cultured tumor cells into mice to analyze tumor response to a candidate compound. It’s important to inject the same number of cells into each mouse, so you need to count the viable cells.

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Figure 2. Excluding dead cells using FSC vs PI
 
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Figure 2. Excluding dead cells using FSC vs PI
Dissociated MDA-MB-468 cells (human epithelial breast adenocarcinoma; ATCC HTB-26) were stained with BD Pharmingen™ Propidium Iodide Staining Solution (Cat. No. 556463). A. Live and dead cells were distinguished and gated based solely on light scatter properties. B. An FSC-H vs PI dot plot of events in the Dead Cell gate shows that, as expected, the majority of these cells are both PI+ and FSC-Hlow, consistent with the loss of membrane integrity and cell shrinkage that occurs as cells die. C. An FSC-H vs PI dot plot of events in the Live Cell gate shows a clear though small population of PI+ cells. This observation demonstrates that light scatter can be a good indicator of cell viability. However, in order to more accurately quantify live and dead cells, it is best to use a viability dye.

A previous tip explained how you can use light scatter as an approximate measure of viability. Dead cells exhibit lower FSC and higher SSC due to cell shrinkage and formation of apoptotic bodies during apoptosis. You can see an example in Figure 2A.

When you need more precise discrimination, or if a fluorescence channel is available, we recommend using a viability stain such as propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) to distinguish live from dead cells. Both dyes are impermeable to healthy cells with intact membranes, but permeate cells with compromised membranes, where they bind to DNA.

Figures 2B and 2C show an FSC vs PI plot of the same cells, gated on dead and live cells (respectively) using light scatter. Figure 2B confirms that most cells in the Dead Cell gate are PI+, confirming that they are dead. But Figure 2C shows a small but clear population of PI+ cells in the Live Cell gate as well. Although light scatter can roughly distinguish live and dead cells, a viability dye helps to do so more accurately.

There are several viability stains to choose from. Although PI is standard, it emits fluorescence into two channels (FL2 and FL3). Alternatively, 7-AAD emits only into FL3. BD Horizon™ Fixable Viability Stains (Cat. No. 564407, 564995, 564996, and 564405) allow you to fix the cells before staining and use them later in downstream applications. Finally, BD Pharmingen™ Calcein AM (Cat. No. 564061) discriminates viable cells based on esterase activity rather than membrane integrity.

Read more about BD fixable and calcein viability dyes »

Events

Meeting – April 18–22, 2015 – Philadelphia, PA
AACR 2015 (American Association for Cancer Research) »

Meeting – May 2–6, 2015 – Pittsburgh, PA
Immunology 2015 (American Association of Immunologists) »

Webinar Replay
Integrating Flow Cytometry Into Education »

BD Accuri™ C6 Personal Flow Cytometry Tour
Thursday, May 14, 2015 – San Diego, CA »
Friday, May 29, 2015 – San Jose, CA »

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