BD Accuri News

 

October 2017


Spotlight

Verifying stem cell markers for Parkinson’s disease clinical trials

Spotlight - Garitaonandia

Ibon Garitaonandia is the director of translational research at the International Stem Cell Corporation in Carlsbad, CA. His paper describing preclinical studies for a Parkinson’s disease clinical trial was a recent BD Accuri News Publication Pick. Dr. Garitaonandia told us why verifying markers is crucial in stem cell preparations and described how they use a BD Accuri™ flow cytometer to do it.
Read the interview »

Application Highlight

A bright dye for every channel

The recent introduction of the BD Horizon Brilliant™ Blue 700 (BB700) dye gives researchers a brighter alternative to PerCP-Cy™5.5 to use in the FL3 channel of the BD Accuri™ C6 Plus personal flow cytometer. This new advanced polymer dye, now available in a broad array of conjugates, can help you detect dimly expressed antigens and rare cell populations.

An important principle of panel design is to pair low-density antigens with brighter fluorochromes for better resolution. BB700 adds a bright fluorochrome in a fluorescence channel where previous options were limited, offering additional flexibility in panel design.

App Highlight - BB700

Consider an example from our recent product information sheet on immuno-oncology applications for the BD Accuri C6 Plus.1 The co-inhibitory receptors PD-1 (CD279) and LAG-3 (CD223) are upregulated on activated T cells. This inhibition mechanism is a normal physiological process to attenuate T-cell activation, but it is also exploited by cancer cells to escape immune response. Both antigen-presenting cells and cancer cells can inhibit T-cell activation by binding PD-1 ligands, such as PD-L1 (CD274), to the PD-1 receptor.

Thus, PD-1 and PD-L1 are important targets for research on new cancer drugs. One research challenge is that both PD-1 and PD-L1 are variably expressed prior to cell activation and may require bright dyes to detect and resolve clearly when expression is low.

App Highlight - Fig 1 - Th
Figure 1. Assessment of human T-cell activation using BB700 vs PerCP-Cy5.5
App Highlight - Fig 1 - Large
Figure 1. Assessment of human T-cell activation using BB700 vs PerCP-Cy5.5

Human PBMCs were stimulated with anti-CD3 and anti-CD28 over 72 hours and harvested at the indicated time points for flow cytometric analysis. Cells were stained with BD Pharmingen™ PE Mouse Anti-Human LAG-3 (CD223) (Cat. No. 565616) and with either BD Horizon BB700 or BD Pharmingen™ PerCP-Cy5.5 Mouse Anti-Human CD279 (PD-1) (Cat. Nos. 566460 and 561273) at the indicated time points. Cells were also stained with BD Via-Probe™ Red Nucleic Acid Stain (Cat. No. 565804) for live/dead cell discrimination, and the two-color dot plots were gated on live lymphocytes (data not shown). Results: Upregulation of both LAG-3 and PD-1 was observed in stimulated cells when the bright dyes PE and BB700, respectively, were used (upper plots). However, PD-1+ cell populations were not fully resolved in samples stained with PerCP-Cy5.5 anti-PD-1.

Figure 1 shows levels of PD-1 and LAG-3 over selected intervals after stimulation of peripheral blood mononuclear cells (PBMCs) with anti-CD3 and anti-CD28. One would expect these activated cells to upregulate both molecules, and as shown in both the top and bottom panels, they did increasingly upregulate LAG-3 (paired with the bright fluorochrome PE) in each time interval. The results for PD-1, however, depended on the panel design. When PD-1 was paired with PerCP-Cy5.5 (lower plots), only marginal upregulation was observed. When PD-1 was paired with BB700 (upper plots), the expected upregulation was clearly detected.

Additional data in the same product information sheet shows that cell staining of PD-L1 with BD Horizon Brilliant™ Blue 515 (BB515) yielded better resolution than FITC. Again, BB515 was designed as a brighter alternative to FITC, yet is detected in the same fluorescence channel (FL1).

In these experiments, PerCP-Cy5.5 and FITC were not bright enough to optimally resolve PD-1 and PD-L1. Of course, these popular fluorochromes are still perfectly satisfactory for many if not most flow cytometry experiments. But to detect antigens expressed at low density, in accordance with best panel design practices, brighter dyes are needed to achieve optimal resolution.


Channel Laser Bright Dye
FL1 Blue BB515
FL2 Blue PE
FL3 Blue BB700
FL4 Red APC

Table 1. A bright dye for every fluorescence channel

With the advent of BB700, there is now a bright dye for every fluorescence channel of BD Accuri™ flow cytometers, as shown in Table 1. Both BB515 and BB700 are significantly brighter than their counterparts, yet exhibit less spillover into neighboring channels. And both are available paired with a wide range of conjugates, giving you unprecedented choice and flexibility in panel design.

Easy to use, simple to maintain and affordable, the BD Accuri C6 Plus personal flow cytometer is equipped with a blue laser, a red laser, two light-scatter detectors and four fluorescence detectors. A compact and transportable design, fixed laser alignment, pre-optimized detector settings and automated instrument QC result in a system that is simple to use. For walkaway convenience, the optional BD CSampler Plus accessory offers automated sampling from 24-tube racks or multiwell plates.

Download our product information sheet on immuno-oncology applications of personal flow cytometry »

Browse BD’s multicolor panel design tools and resources »


 

Tips & Tricks

The Zoom tool

Tips and Tricks - Zoom
Accuri News - Figure 3 - Thumb
Figure 2. Use the Zoom tool to locate populations
 
Accuri News - Figure 3 - Large
Figure 2. Use the Zoom tool to locate populations
Human blood cells were acquired and analyzed for light scatter on the BD Accuri™ C6 system. A. FSC vs SSC plot at original scale, with a wide zoom area drawn around several cell populations including lymphocytes. B. The zoomed plot, with a new, narrower zoom area drawn around the lymphocyte and red blood cell populations. C. After zooming twice, the lymphocytes are easily recognized. D. A gate is drawn around the lymphocytes. E. When the plot is zoomed out again, the gate remains in place.

With the broad dynamic range of BD Accuri systems, it is often useful to focus in on a particular subset of channels or events. The Zoom tool provides such focus and is indispensable for locating cell populations and drawing accurate gates.

In Figure 2, an FSC vs SSC plot of a whole blood sample, you might find it difficult to locate the lymphocyte population. To zoom in, click the Zoom tool and draw a wide region that includes the lymphocyte population. Repeat as needed, drawing progressively smaller regions until you can easily identify the lymphocytes and draw an accurate gate. Then zoom back out again using the Expand tool.

Accuri News - Figure 4 - Thumb
Figure 3. Use the Zoom tool to adjust gates
 
Accuri News - Figure 4 - Large
Figure 3. Use the Zoom tool to adjust gates
Human blood cells were stained and analyzed on the BD Accuri C6. A. Original polygon placement. B. Zoomed plot shows overlapping polygons. C. Adjusted polygons. D. Plot zoomed back to original scale. For method details, see the BD Biosciences technical bulletin, Identification of Human Peripheral Blood Cell Populations with the BD Accuri™ C6 Flow Cytometer (November 2011), available at bdbiosciences.com.

In Figure 3, whole human blood was immunophenotyped with fluorescent stains to identify five cell populations. Backgating was used to place all five populations on a CD45 vs SSC plot, but the gates were not precise and the polygons overlapped. By zooming in on the plot, the polygons can be adjusted to avoid overlap. The Zoom tool allows precise control when setting gates.


 

Publication Picks

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

Nanotherapy for Parkinson’s disease

Wang N, Jin X, Guo D, Tong G, Zhu X. Iron chelation nanoparticles with delayed saturation as an effective therapy for Parkinson disease. Biomacromolecules. 2016;18:461-474. PubMed

Pancreatitis treatment

Wang, GJ, Wang Y, Teng YS, et al. Protective effects of emodin-induced neutrophil apoptosis via the Ca2+-caspase 12 pathway against SIRS in rats with severe acute pancreatitis. Biomed Res Int. 2016;2016:1736024. PubMed

Methods for in vitro algal growth

Huesemann M, Dale T, Chavis A, et al. Simulation of outdoor pond cultures using indoor LED-lighted and temperature-controlled raceway ponds and Phenometrics photobioreactors. Algal Research. 2017;21:178-190. Full text

Sperm testing

Zhang G, Wang Z, Ling X, et al. Mitochondrial biomarkers reflect semen quality: Results from the MARCHS study in Chongqing, China. PloS One. 2016;11:e0168823. 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.