BD Accuri News

 

February 2018


Spotlight

New methods of generating neuronal cells from stem cells

Spotlight - Hong

Yiling Hong is associate professor at the Western University of Health Sciences in Pomona, CA. Her team’s paper on optimal methods for generating neuronal cells from embryonic stem cells and induced pluripotent stem cells (iPSCs) was a recent BD Accuri News Publication Pick. Dr. Hong explained the role of her BD Accuri™ flow cytometer in monitoring pluripotency and differentiation, and discussed both nanoparticles and spicy foods.
Read the interview »

Noteworthy

BD acquires FlowJo®

Noteworthy - Flow to tha Jo

We’re pleased to announce that BD has acquired FlowJo, LLC, which makes popular software for analysis and interpretation of flow cytometric and genomic data. FlowJo is now a wholly owned subsidiary of BD but will operate as a separate company to ensure minimal disruption to customers and partners. FlowJo® software will continue to support analysis of data acquired from all flow cytometers, regardless of manufacturer.
Learn more »

Application Highlight

How flow cytometry can complement conventional methods in cell and molecular biology

If you’re a cell or molecular biologist, you’re always looking for ways to enrich the data you collect from your samples. Flow cytometry can complement other methods in your toolbox by providing a quantitative analysis of protein expression in cell-based models. On the BD Accuri™ C6 Plus personal flow cytometer, you can run these studies right on your benchtop.

Suppose we’re investigating the differentiation and gene revelation of pluripotent human embryonic stem cells (hESCs) into definitive endoderm (Figure 1). Upon differentiation, we expect lineage-specific genes to be activated, and we can assess activation by analyzing the expression of FoxA2 and Sox17.


App Highlight - Fig 1

Figure 1. Differentiation of hESCs into definitive endoderm
In the presence of low fetal bovine serum (FBS) and activin, hESCs differentiate into a heterogeneous population consisting of definitive endoderm and other cells.


Figure 2 shows three ways of looking at this process. Conventional Western blot analysis (Figure 2A) shows that, by 72 hours, the cells have differentiated and the FoxA2 and Sox17 genes have been upregulated, as demonstrated by the expression of the lineage-specific proteins. However, we can’t tell whether differentiation has occurred homogeneously in all the cells expressing small amounts of the proteins, or in a subset of cells expressing large amounts. Western blot is a bulk analysis on a total cell lysate.

App Highlight - Fig 2 - Th

Figure 2. Three methods of assessing protein expression in cell-based models

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Figure 2. Three methods of assessing protein expression in cell-based models

hESCs were cultured in the presence of low FBS and activin for 72 hours, as illustrated in Figure 1, to induce differentiation into definitive endoderm. FoxA2 and Sox17 expression was evaluated using three methods: (A) Western blot, (B) fluorescence microscopy and (C) flow cytometry. Flow cytometry provided a precise quantitative assessment of the percentage of cells expressing both markers.

We can also look at the process using microscopy (imaging), another methodology used conventionally in cell biology and molecular biology laboratories. Imaging can give us a better idea of the percentage of cells that have differentiated. In Figure 2B, we can see which cells are expressing the same markers, FoxA2 (yellow) and Sox17 (green), after 72 hours, corroborating the data from Western blot. Still, it would be difficult to determine how many cells have gone through this process.

Finally, Figure 2C shows how flow cytometry can quantify expression of the same antibodies and markers. Consistent with other methods, the results confirm that after 72 hours, the genes have been activated and the proteins are being expressed. But now we can also determine precisely the frequency of cells—57%—that are positive for both markers. This is because flow cytometry provides a quantitative, single-cell analysis.

A flow cytometer is an essential cell analysis tool for every cell and molecular biology lab. With the ability to analyze large, complex populations at the single-cell level, multiparametric flow cytometry provides a perspective on cell process and function that other methodologies cannot.

Versatile, compact, and affordable, the BD Accuri C6 Plus brings the power of flow cytometry within reach for individual research labs and small institutions. With its combination of performance and simplicity, the BD Accuri C6 Plus puts this essential cell analysis tool into the hands of more cell and molecular biologists than ever before.

Download our white paper, The Essential Cell Analysis Tool »


 

Tips & Tricks

Reveal hidden data

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Figure 3. Two ways to reveal “hidden” data

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Figure 3. Two ways to reveal “hidden” data

Instrument Setup Beads from the BD™ Cytometric Bead Array (CBA) Human Soluble Protein Master Buffer Kit (Cat. No. 558264 or 558265) were collected on the BD Accuri C6 and gated using either (A–C) region gates or (D–E) quadrant gates. A. Uncompensated, 56.9% of the CBA beads fall in R2 and 42.5% in R3. B. After compensation is applied, beads shift out of the region gates (37.3% in R2 and 29.5% in R3). C. By displaying the first decade, the region gates are extended to the origin and now include all relevant events, including some that were uncounted before compensation. Percentages now total 100%. D, E. Using quadrant gates instead of region gates, all of the beads are included in the analysis, both (D) without compensation and (E) with compensation.

By default, the first decade of data (MFI = 1–10) is omitted from plots in BD Accuri™ software. Although this default setting (implemented at customer request) is designed to mask only noise, very dim populations may be hidden as well.

Accuri News - June - Figure 3 - Thumb

Figure 4. Displaying the first decade of data

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Figure 4. Displaying the first decade of data

To display the first decade of data, (A) click Plot Spec below the plot. In the dialog box that appears, (B) deselect Hide 1st decade for both axes and click Apply to accept the changes.

This can sometimes lead to confusion when performing fluorescence compensation. When data is compensated, events can be moved into this decade (or even to channel 1), making it appear as if the data is missing or lost. The data shift may also affect statistical calculations, such as percentages, if regions don't extend all the way to the first channel.

The missing data is not gone, only hidden. Figure 3 shows two ways of revealing it. One is to change the Hide 1st decade setting to display channels 1–10, as shown in Figure 4. The second is to use quadrant gates instead of region gates, or to draw the regions to extend all the way to channel 1.

Note that the software does not currently save the Hide 1st decade setting. If you use this technique, you will need to change the setting again when you reopen the file.


 

Publication Picks

This section highlights interesting recent articles that describe research using BD Accuri flow cytometers.

Preventing infection in burn victims

Patil NK, Liming K, Bohannon JK, Hernandez A, Guo Y, Sherwood ER. Anti-PD-L1 protects against infection with common bacterial pathogens after burn injury. J Leukoc Biol. 2017;1-11:doi: 10.1002/JLB.5HI0917-360R. Full text

Zika virus

Ricciardi MJ, Magnani DM, Grifoni A, et al. Ontogeny of the B- and T-cell response in a primary Zika virus infection of a dengue-naïve individual during the 2016 outbreak in Miami, FL. PLoS Negl Trop Dis. 2017;11:e0006000. PubMed

Bacteria in pool water

Peters MCFM, Keuten MGA, Knezev A, et al. Characterization of the bacterial community in shower water before and after chlorination. J Water Health. 2017;doi: 10.2166/wh.2017.189. Abstract

Gold nanoparticles as cancer therapeutics

Reddy TS, Privér SH, Mirzadeh N, Bhargava SK. Anti-cancer gold(I) phosphine complexes: Cyclic trimers and tetramers containing the P-Au-P moiety. J Inorg Biochem. 2017;175:1-8. PubMed


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1 All reagents and kits are compatible with both the BD Accuri C6 Plus and the BD Accuri C6 flow cytometer systems. Data was generated on the BD Accuri C6 Plus. Information about BD reagent kits, BD Accuri™ C6 and BD Accuri C6 Plus software templates, and BD CSampler™ and BD CSampler Plus automation options is available at www.bdbiosciences.com/accuri.