BD ACCURI NEWS

 

August 2018


Noteworthy

New FlowJo® release

Noteworthy - Flowjo Release

If you use software from FlowJo® (now a subsidiary of BD) to analyze your flow cytometry data, check out the new release. With FlowJo v10.5, you can now purchase and manage your license through the FlowJo Portal. Once you create your free account, you can use your FlowJo Portal ID to sign in to FlowJo anywhere, at any computer—no dongle or hardware address required. Learn more »

Application Highlight

Measuring rituximab-mediated complement-dependent cytotoxicity

The therapeutic efficacy of anti-tumor monoclonal antibodies depends on their capacity to recognize the tumor-associated antigen and trigger immune defense mechanisms against the tumor. Rituximab is a chimeric murine/human monoclonal antibody (mAb) targeting CD20, a cell surface antigen expressed primarily by most B cells, which makes it a potent cancer therapeutic to treat a broad variety of CD20-positive malignances and B-cell disorders.

Rituximab-mediated B-cell cytotoxicity is driven in part by activation of proteins from the complement system, leading to the formation of the membrane attack complex (MAC) and subsequent cytolysis of the target cell. Thus, the efficacy of rituximab complement-dependent cytotoxicity (CDC) depends on CD20 antigen density.

A new BD data sheet shows how the BD Accuri™ C6 Plus personal flow cytometer can measure CD20 antigen density and receptor occupancy by rituximab. We also tested whether rituximab induced the lysis of CD20-expressing cells in the presence of normal human serum. The BD Accuri C6 Plus can count cells volumetrically, enabling assessment of cell viability by analyzing absolute cell numbers instead of counting beads.1

App Highlight - Fig 1

Figure 1. Rituximab-mediated CDC against Daudi lymphoma cells

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Figure 1. Rituximab-mediated CDC against Daudi lymphoma cells

Daudi cells (Burkitt’s lymphoma, ATCC-CCL-213) were maintained in media containing heat-inactivated fetal bovine serum (56°C for 30 min). The cells were counted using the BD Accuri C6 Plus flow cytometer system, resuspended at 1x106 cells/mL and 100 μL of cells were transferred to the wells of a 96-well U-bottom plate. Serial twofold dilutions (ranging from 0.1 μg/mL to 20 μg/mL) of the monoclonal therapeutic antibody rituximab (BioVision) or human IgG1 isotype control (BioLegend) were added to the cells. Cell cultures were supplemented with 25% normal or heat-inactivated human serum was added to the cells and the plates were incubated at 37°C for 5 hours. The plates were spun down and the cells resuspended in 10% BD Pharmingen™ 7-AAD solution. Results: The graph shows the concentration of live 7-AAD cells in the samples, measured by the BD Accuri C6 Plus as number of events/μL. Rituximab-induced dose-dependent lysis of Daudi cells in the presence of normal human serum was demonstrated by the reduction in live 7-AAD cells (blue curve). This effect was inhibited upon heat-inactivation of the serum (orange curve), suggesting a role for the complement in rituximab-mediated cell death.

Figure 1 shows that rituximab induced the lysis of CD20-expressing cells in a dose-dependent manner. Incubation of Daudi (Burkitt’s lymphoma) cells with rituximab and normal human serum led to rapid cell death. The number of live (7-AAD) Daudi cells decreased upon treatment with rituximab and normal human serum, but not when the serum was heat-inactivated, suggesting the involvement of temperature-sensitive components such as complement in tumor cell killing.

For the receptor-occupancy assay, we identified a CD20 antibody, PE-L27, that competed with rituximab for binding to CD20. This fluorochrome-conjugated antibody was then used to estimate the number of rituximab molecules binding to the target cells (Figure 2A).

App Highlight - Fig 2

Figure 2. Rituximab receptor occupancy and Daudi-cell cytolysis

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Figure 2. Rituximab receptor occupancy and Daudi-cell cytolysis

Daudi cells were counted using the BD Accuri C6 Plus and 1x105 cells were incubated with twofold serial dilutions of rituximab (ranging from 0.05 μg/mL to 20 μg/mL) at 37°C for 30 min. The cells were washed and stained with PE Mouse Anti-Human CD20, clone L27 (PE-L27), which competes with rituximab for binding to CD20, as previously determined (data not shown). In parallel, the BD Quantibrite™ PE Phycoerythrin Fluorescence Quantitation kit was used to estimate the number of anti-CD20 molecules bound per Daudi cell. Additionally, rituximab-coated cells were cultured at 37°C for 5 hours in the presence of 25% normal human serum to analyze cell viability. A. The numbers of anti-CD20 (PE-L27) bound per cell appear gray, while the calculated numbers of CD20 receptors occupied by rituximab appear blue in the graph. B. The graph shows a correlation between CD20 receptor occupancy and cell viability. The blue curve represents the percentages of cells with CD20 receptors occupied by rituximab, while the orange curve represents the percentages of 7-AAD live cells relative to cells cultured without rituximab (100% cell viability). Results: The BD Quantibrite PE Phycoerythrin Fluorescence Quantitation kit combined with PE-L27 enabled the calculation of the total numbers of anti-CD20 bound per cell and the CD20 receptors bound to rituximab. Cell viability decreased in proportion to receptor occupancy, demonstrating that the level of cell surface CD20 is a critical factor in rituximab-driven target cell lysis.

First, Daudi cells were stained with PE-L27 only and the total number of CD20 receptors expressed per cell was measured (vertical bars). Separately, the cells were incubated with increasing doses of rituximab followed by staining with PE-L27. In this case, only CD20 receptors unoccupied by rituximab were available for binding by the PE-L27 antibody (gray bar segments). The number of rituximab-occupied receptors (blue bar segments) was then calculated by subtracting the level of unoccupied CD20 receptors from total CD20 expression. Figure 2B shows the strong inverse correlation between rituximab occupancy and cell viability.

Lastly, as detailed in the data sheet, we analyzed rituximab-mediated and complement-dependent B-cell killing using peripheral blood mononuclear cells. The volumetric counting capability of the BD Accuri C6 Plus was used to determine the absolute numbers of CD19+ B-cell and CD3+ T-cell subsets.

The experiments in the new data sheet demonstrate the ability of flow cytometry to determine antigen density, receptor occupancy and antibody-mediated B-cell killing. Affordable, compact and easy to use, the BD Accuri C6 Plus system enables research labs to perform these experiments—and many others—on their benchtop and in the field.

Download the new data sheet »
Download the BD Accuri C6 brochure »
Visit the BD Accuri Cell & Cancer Biology page »


 

Tips & Tricks

When to use optional filters

What can you do if a multicolor experiment includes two fluorochromes with overlapping emission spectra? In some cases, you can simply substitute another fluorochrome that emits into a different detector. But suppose your cells are tagged with both green and yellow fluorescent proteins (GFP and YFP) and you need to discriminate them?

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Figure 3. How can you detect GFP and YFP simultaneously?

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Figure 3. How can you detect GFP and YFP simultaneously?

A, C. Either GFP or YFP signals can be detected in FL1 and FL2 with the standard BD Accuri™ filter configuration (533/30 in FL1, 585/40 in FL2). B, D. To detect both GFP and YFP at once, separate the signals by using the 510/15 filter (Cat. No. 653184) in FL1 and the 540/20 filter (Cat. No. 653528) in FL2. The top and bottom plots show uncompensated and compensated data, respectively.

Figure 3 illustrates the problem: a mixed population in which some cells express GFP, some YFP and some neither. Using the standard filter configuration of a BD Accuri™ flow cytometer, before compensation (Figure 3A), these two proteins emit fluorescence into both the FL1 and FL2 channels. Compensation (Figure 3B) merely eliminates most of the FL2 signal, so that both appear primarily in FL1. Although you could easily detect either of these fluorochromes separately, it is impossible to differentiate one from the other.

App Highlight - Fig 4

Figure 4. BD Accuri optical bench with standard filters

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Figure 4. BD Accuri optical bench with standard filters

To separate the signals, you can use optional filters instead of the standard ones. To detect GFP in the FL1 detector, substitute the optional 510/15 BP filter (Cat. No. 653184) for the standard 533/30 BP filter. To detect YFP in the FL2 detector, substitute the optional 540/20 BP filter (Cat. No. 653528) for the standard 585/40 BP filter. Figures 3C and 3D show that GFP and YFP signals are now separated and, after compensation, each can be detected in its own channel.

Filters in BD Accuri systems are designed to be easily interchangeable on the optical bench (Figure 4). Simply slide out the standard filter and slide in the optional one. It’s just as easy to restore the standard configuration when you’re done. Keep optional filters in mind when you want to increase signal resolution or to separate signals that might otherwise overlap.

Download the Optical Filter Guide »
Download the technical bulletin, Optical Filter Guidelines for Fluorescent Protein Analysis »


 

Publication Picks

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

Biofilms in shower hoses

Proctor CR, Reimann M, Vriens B, Hammes F. Biofilms in shower hoses. Water Res. 2017;131:274-286. PubMed

Isoginkgetin effects on cell cycle progression

Vanzyl EJ, Rick KRC, Blackmore AB, MacFarlane EM, McKay BC. Flow cytometric analysis identifies changes in S and M phases as novel cell cycle alterations induced by the splicing inhibitor isoginkgetin. PLoS One. 2018;13:e0191178. PubMed

New peptide protects post-transplant stem cells

Park HH, Lee KY, Park DW, et al. Tracking and protection of transplanted stem cells using a ferrocenecarboxylic acid-conjugated peptide that mimics hTERT. Biomaterials. 2018;155:08-91. PubMed

Phytoplankton evolution

Bach LT, Lohbeck KT, Reusch TBH, Riebesell U. Rapid evolution of highly variable competitive abilities in a key phytoplankton species. Nat Ecol Evol. 2018;2:611-613. PubMed


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.

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