BD ACCURI NEWS

 

December 2017


Spotlight

Studying microorganisms in their natural plant habitats

Spotlight - Leveau

Johan Leveau is a professor of plant pathology at the University of California, Davis, where his research interests include individual-based microbial ecology. His co-authored paper on bacterial symplasmata was a recent BD Accuri News Publication Pick. Dr. Leveau told us why green fluorescent protein (GFP) is a “wonder tool” for getting a microbe’s take on its local environment, and how his BD Accuri™ flow cytometer helps in this research.
Read the interview »

Noteworthy

Choose up to three free options with a new BD FACSCelesta™ flow cytometer

Noteworthy - Celesta

Accurate. Efficient. Proven. With up to three lasers and 14 parameters, the BD FACSCelesta™ flow cytometer brings together bright new polymer dyes and innovative software technologies in a single system. For a limited time, pick up to three of the following options—a BD™ High Throughput Sampler (HTS), a BD FACSFlow™ Supply System (FFSS), reagent credits or an additional year of warranty service—free with purchase of a new BD FACSCelesta system.
Learn more »

Application Highlight

Assessing CD8+ cytotoxic T-cell activation and response

In the field of immuno-oncology, extensive studies have investigated how cytotoxic T cells can overcome tumor-induced immunosuppressive signals and become activated. Activated T cells proliferate, secrete pro-inflammatory cytokines and release lytic granules (degranulation). The granules contain proteins such as perforin and granzymes, which may cause irreversible damage to tumor cells.

A new BD product information sheet shows how the multiplexing capability of multicolor flow cytometry enables simultaneous analyses of these cellular processes in CD8+ cytotoxic T cells at the single-cell level. On the BD Accuri™ C6 Plus flow cytometer, with two lasers and four fluorescence parameters, you can assess these activation measures right on your benchtop. This resource complements a similar analysis of NK cell activation and cytotoxicity as discussed in our September 2017

The experimental model, shown in Figure 1, employs a one-way mixed lymphocyte reaction (MLR) assay to evaluate CD8+ T-cell-specific response against alloantigens. Besides providing insights into antigen-specific response, the MLR assay can provide a multifaceted window into T-cell function, including proliferation, degranulation and cytokine production. In this assay, responder CD3+ T cells from one donor were magnetically enriched from PBMCs and co-cultured with T cell-depleted stimulator cells, either from the same donor (autologous) or a different donor (allogeneic). Responder cells were labeled with CFSE to measure cell proliferation as well as to facilitate separation from CFSE stimulator cells.


App Highlight - Fig 1

Figure 1. One-way MLR assay to evaluate CD8+ T-cell response
Responder T cells from donor A are co-cultured with stimulator cells from either the same donor A (autologous) or a different donor B (allogeneic). T-cell responses include proliferation (measured using CFSE dilution), degranulation (exposure of CD107a on the cell surface) and cytokine production (intracellular IFN-γ expression).


App Highlight - Fig 2 - Th
Figure 2. Proliferation of CD8+ T cells in an MLR assay on the BD Accuri C6 Plus
App Highlight - Fig 2 - Large
Figure 2. Proliferation of CD8+ T cells in an MLR assay on the BD Accuri C6 Plus

CFSE-labeled T cells were cultured with allogeneic or autologous mitomycin C-treated stimulator cells and stained for analysis on the BD Accuri C6 Plus. Unstimulated cells and cells treated with SEB were used as negative and positive controls, respectively. Cells were gated on CD3+ CFSE+/low responder T cells after exclusion of CFSE stimulators (not shown). Results: Analysis of CFSE dilution showed that allogeneic stimulation induced modest, alloantigen-specific proliferation (CFSElow) of CD3+ T cells in the MLR cultures, as compared to SEB. As expected, no cell proliferation was observed upon autologous stimulation, similar to unstimulated cells. Further analysis (shown in product information sheet) showed surface exposure of CD107a and IFN-γ production, indicating degranulation and activation, among proliferating CD8+ T cells.

We designed two 4-color panels for the simultaneous analysis of CFSE, CD3, CD8 and either CD107a or IFN-γ. Figure 2 shows the analysis of CFSE, which dilutes with each successive generation, thus indicating proliferation. Results showed a modest increase in T-cell proliferation (CFSElow cells) in response to allogeneic stimulation as compared to SEB stimulation, the positive control. The response was specific to alloantigens, as stimulation with autologous stimulator cells caused minimal proliferation, similar to unstimulated cells, the negative control. Further analysis (shown in the product information sheet) showed that allogeneic stimulation also induced degranulation (shown by CD107a exposure on the cell surface) and cytokine production (shown by expression of intracellular IFN-γ) by proliferating CD8+ T cells, indicating that these cells were fully activated.1

The use of flow cytometry to assess T-cell activation allows you to answer important immuno-oncological questions. The BD Accuri C6 Plus personal flow cytometer can assess immune cell proliferation and activation simultaneously on a benchtop.

Download the product information sheet »

Check out our BD Accuri C6 Plus promotion »


 

Tips & Tricks

Positive controls

Positive controls can be helpful in achieving optimal results on flow cytometry assays. The compounds camptothecin and staurosporine, for example, are commonly used to induce apoptosis in cell lines, while aphidicolin is often used as a positive control to induce cell cycle arrest. In this month’s Application Highlight, SEB stimulation was used to induce maximum proliferation among T cells.

Prior to analyzing precious experimental samples, calculate an initial dose-response curve with an established positive control compound for your assay. The results can help you determine a gating strategy and define assay criteria.


 

Publication Picks

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

Impact of fatty acids on immunity

Passos ME, Alves HH, Momesso CM, et al. Differential effects of palmitoleic acid on human lymphocyte proliferation and function. Lipids Health Dis. 2016;15:217. PubMed

Silver nanoparticle development

Orlov IA, Sankova TP, Babich PS, et al. New silver nanoparticles induce apoptosis-like process in E. coli and interfere with mammalian copper metabolism. Int J Nanomedicine. 2016;11:6561-6574. PubMed

Chikungunya vaccine development

Erasmus JH, Auguste AJ, Kaelber JT, et al. A chikungunya fever vaccine utilizing an insect-specific virus platform. Nat Med. 2017;23:192-199. PubMed

Safety of green tea extract

Peluso I, Manafikhi H, Raguzzini A, et al. The peroxidation of leukocytes index ratio reveals the prooxidant effect of green tea extract. Oxid Med Cell Longev. 2016;2016:9139731. 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.