BD ACCURI NEWS

 

April 2017


Spotlight

The BD FACSCelesta™ flow cytometer in the core facility

Spotlight - Daniel Interview

Benjamin J. Daniel is the director of the Institutional Flow Cytometry Facility and a Research Assistant Professor at the UT Health, San Antonio, TX. He is also Vice President of FlowTex, a Texas-based annual flow cytometry conference. Dr. Daniel told us why a new 3-laser BD FACSCelesta™ flow cytometer quickly became popular at his core facility, and described the various research groups that use it.
Read the interview »

Noteworthy

Unbelievable savings on the BD Accuri C6 Plus

Noteworthy - Accuri Promo Thumb

Easy to use. Versatile. Affordable. Unlock your understanding of cells and cell populations with the BD Accuri™ C6 Plus personal flow cytometer. For a limited time, take advantage of unbelievable savings: get a 35% discount on the purchase of a BD Accuri C6 Plus—or a 40% discount on the BD Accuri C6 Plus with the BD CSampler™ Plus automation accessory.
Learn more »

BD Accuri videos now online

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The BD Accuri C6 Plus hits the big screen—or at least the small one! You can now view an overview of the instrument, as well as instructional videos covering installation and each tab of BD Accuri™ C6 Plus software. You’ll also find them on our YouTube channel. Settle back and click Play—all you need to bring is popcorn!
View the overview video »
View the instructional videos »

Application Highlight

Immuno-oncology applications of personal flow cytometry

Immuno-oncology has become the subject of intense scientific and therapeutic interest, as researchers probe the mechanisms by which cancer cells inhibit T-cell activation and escape T-cell attack, and explore methods of circumventing this inhibition. Because these research questions are inherently multivariate, involving the complementary push-and-pull signaling of activation and inhibition, they are ideal for multicolor flow cytometry, which can assess multiple parameters in heterogeneous cell populations at the single-cell level.

A new BD data sheet explores the benefits of flow cytometry for immuno-oncology studies. On the BD Accuri C6 Plus personal flow cytometer, you can perform these studies right on your benchtop.

Co-inhibitory receptors on immune cells are crucial regulators of T-cell and dendritic cell activation. This inhibition mechanism is a normal physiological process to attenuate T-cell activation, but cancer cells also exploit it to escape immune response. For example, the PD-1 (CD279) receptor is upregulated on activated T cells. 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 PD-L1 Thumb
Figure 1. PD-L1 upregulation in cancer cells following different stimulation treatments
App Highlight - Fig 1 PD-L1
Figure 1. PD-L1 upregulation in cancer cells following different stimulation treatments

A. MDA-MB-468 breast cancer cells were stimulated with BD Pharmingen™ rhIFN-γ (Cat. No. 554617) and stained with either BD Horizon BB515 or BD Pharmingen™ FITC Anti-Human CD274 (PD-L1) (Cat. Nos. 564554 and 558065). BB515 staining (blue) resulted in stronger fluorescence signal and better separation between stained and unstained cells. B. Robust upregulation of PD-L1 was observed when cells were cultured overnight with either conditioned medium from stimulated PBMCs (24h supernatant, red) or a combination of the recombinant cytokines IFN-γ and BD Pharmingen™ rhTNF (Cat. No. 554618, blue), whereas very low levels of PD-L1 expression were detected in cells cultured with supernatants from unstimulated PBMCs (green).

The cytokines IFN-γ and TNF are known to increase the expression levels of PD-1 ligands in antigen-presenting cells as well as in certain tumor cells. These ligands can be assessed on cancer or antigen-presenting cells using flow cytometry. Figure 1B demonstrates that either conditioned medium from activated peripheral blood mononuclear cells (PBMCs) (red) or a combination of rhIFN-γ and rhTNF cytokines (blue) induced PD-L1 expression in the MDA-MB-468 breast-cancer cell line. A similar induction was not observed when treating the cells with conditioned medium from unstimulated PBMCs (green).1

Note that, in Figure 1A, cell staining with BD Horizon Brilliant™ Blue 515 (BB515) PD-L1 yielded better resolution than FITC PD-L1. BB515 was designed as a brighter alternative to FITC, yet is detected in the same fluorescence channel (FL1).

App Highlight - Fig 2 PD-L1 CD83 Thumb
Figure 2. PD-L1 and CD83 expression following induction and blockage of DC maturation
App Highlight - Fig 2 PD-L1 CD83
Figure 2. PD-L1 and CD83 expression following induction and blockage of DC maturation

Human monocyte-derived DCs were differentiated using GM-CSF and IL-4. A. Immature DCs obtained on day 6 of culture did not express CD83 or PD-L1 surface markers. B–C. Upregulation of these markers was observed upon maturation induced by 3-day stimulation with rhIFN-γ and rhTNF or conditioned medium (24h supernatant) from activated PBMC cultures. D. Cells were also stimulated with the same conditioned medium in the presence of anti-IFN-γ and anti-TNF blocking antibodies, which resulted in a significant inhibition of maturation as indicated by the lack of CD83 and PD-L1 expression.

Figure 2 shows that stimulation of immature dendritic cells (iDCs, involved in tumor antigen presentation) with either conditioned medium (24h supernatant, plot C) or recombinant cytokines (rhIFN-γ and rhTNF, plot B) resulted in both cell maturation (indicated by upregulation of CD83) and induction of PD-L1. However, adding anti-IFN-γ and anti-TNF blocking antibodies to the conditioned medium prevented upregulation of CD83 and PD-L1, and ultimately maturation of the DCs (plot D).

The data sheet contains additional examples showing assessment of the receptors PD-1 and LAG-3 (CD223) on activated T cells, and the use of BD™ Cytometric Bead Array (CBA) for quantifying cytokines.

A personal flow cytometer in the lab provides many advantages for cell and cancer biology studies. When cells are ready for analysis or rare tumor samples arrive, it’s crucial to have a flow cytometer at hand, ready to go.

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 the data sheet »

Download the BD Accuri C6 Plus brochure »

Visit the BD Accuri Immunology page »


 

Tips & Tricks

New panel design tool: BD Horizon™ GPS

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Figure 3. Improving panel design with BD Horizon GPS
Tips Tricks - Fig 3 GPS
Figure 3. Improving panel design with BD Horizon GPS

For three iterations of a sample panel design, the figure shows typical BD Horizon GPS alerts and sample data.

Note: This six-color sample panel would not be appropriate for a four-color BD Accuri cytometer.

Good panel design is essential to obtaining good data. The BD Horizon™ Guided Panel Solution (GPS) provides a guided interactive workflow for reagent selection based on the principles of panel design. This free online tool—currently in beta test release—will help you streamline the panel design process and avoid reagent selections that may negatively affect population resolution.

When using the tool, you will be prompted to input information about your panel. The tool already has knowledge about fluorochrome brightness, instrument configurations and resolution impact, so you don’t have to enter that information. It takes all of this information into account to alert you to reagent selections that might reduce population resolution.

To use BD Horizon GPS, you will need a free account on bdbiosciences.com. This allows you to save and manage panels within your account. All documentation is provided online.

Try out BD Horizon GPS »


 

Publication Picks

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

Natural cancer therapies

Dia V, Krishnan HB. BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells. Sci Rep. 2016;6:33532. PubMed

Bacterial behavior

Tecon R, Leveau JHJ. Symplasmata are a clonal, conditional, and reversible type of bacterial multicellularity. Sci Rep. 2016;6:31914. PubMed

Nanotherapy

Xia L, Gu W, Zhang M, et al. Endocytosed nanoparticles hold endosomes and stimulate binucleated cells formation. Part Fibre Toxicol. 2016;13:63. PubMed

Probiotics

Xing Z, Tang W, Geng W, Zheng Y, Wang Y. In vitro and in vivo evaluation of the probiotic attributes of Lactobacillus kefiranofaciens XL10 isolated from Tibetan kefir grain. Appl Microbiol Biotechnol. 2016;101:2467-2477. PubMed


 

Events

Meeting – May 12–16, 2017 – Washington, DC
Immunology 2017 (American Association of Immunologists) »

Meeting – June 10–14, 2017 – Boston, MA
CYTO 2017 (International Society for Advancement of Cytometry) »

Meeting – June 14–17, 2017 – Boston, MA
ISSCR 2017 (International Society for Stem Cell Research) »


Class 1 Laser Product.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.

1 All reagents and kits are compatible with both the BD Accuri C6 and the new BD Accuri C6 Plus 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.