BD Accuri News

 

July 2018


Application Highlight

A flow cytometric assay to assess phagocytosis modulation

Phagocytosis by macrophages is one of the innate immune system’s first line of defense mechanisms against pathogens and transformed cells. CD47, expressed by all normal cells, is a critical regulator of phagocytosis. By binding to the ligand signal regulatory protein α (SIRP-α) on macrophages, it can prevent phagocytosis of healthy cells and regulate self-tolerance.

This inhibitory mechanism is exploited by tumor cells that express high levels of CD47. The observation that blocking CD47:SIRP-α interaction enables increased phagocytosis of tumor cells has led to several clinical trials based on blocking CD47 through therapeutic monoclonal antibodies. However, high variability in response to CD47 blockade across cancer cell lines suggests that there may also be alternative inhibitory mechanisms.

A new BD data sheet demonstrates how both CD47 blocking and phagocytosis can be assessed on your benchtop using the BD Accuri™ C6 Plus personal flow cytometer.1 Based on a simple, rapid screen of numerous cancer cell lines, we selected three cell lines that differed in CD47 expression. Cell line 697 (acute lymphoblastic leukemia) expresses moderate levels of CD47. In contrast, the HepG2 (human liver cancer) and Caco-2 (human epithelial colorectal cancer) cell lines express extremely low levels of CD47.

App Highlight - Fig 1
Figure 1. Validation of CD47 blocking
App Highlight - Fig 1 Zoom
Figure 1. Validation of CD47 blocking

The 697 cells were incubated for 30 minutes at room temperature with 10 µg of BD Pharmingen™ Purified NA/LE Mouse Anti-Human CD47, prior to staining with BD Pharmingen™ FITC Mouse Anti-Human CD47 (red, A). Cells stained with FITC anti-CD47 alone (B), or cells pre-incubated with BD Pharmingen™ Purified Mouse IgG1 (isotype control), followed by staining with FITC-CD47 (C), were used as controls. Results: Incubation with purified NA/LE anti-CD47 resulted in complete blocking of CD47 as compared to controls.

Figure 1 demonstrates that 10 μg of anti-CD47 blocking antibody (purified NA/LE CD47) binds to all CD47 receptors available on the cell surface. We incubated the cells first with the blocking antibody followed by staining with the same antibody clone conjugated to FITC. The lack of FITC signal confirmed that all anti-CD47 binding sites were indeed occupied by the blocking antibody.

We then co-cultured the different CFSE-labeled cancer cell lines with monocyte-derived macrophages and analyzed the cells on the BD Accuri C6 Plus. Labeling the target cells with CFSE allowed us to measure phagocytosis by detecting CFSE uptake in the macrophage population, identified by their CD11b expression (CD11b+CFSE+). Due to the brightness of CFSE, we used the optional 99% attenuation filter in FL1 to bring the signal onto scale.

App Highlight - Fig 2
Figure 2. Effects of blocking CD47 on phagocytosis
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Figure 2. Effects of blocking CD47 on phagocytosis

Monocyte-derived macrophages were co-cultured with CFSE-labeled 697 (A), HepG2 (B) or Caco-2 (C) cells at a 1:1 ratio along with 10 µg of either purified mIgG1 (left plots) or purified NA/LE mouse anti-human CD47 (right plots) in a U-bottom 96-well plate. Cells were stained with CD11b APC to identify the macrophages. Cells were spun down at 300xg and co-cultured for 6 hours. Samples were acquired on the BD Accuri C6 Plus fitted with the BD Accuri™ FL1 99% Attenuation Filter. Results: Macrophages and target cancer cell lines can be discriminated based on the mutually exclusive expression of CD11b and CFSE, respectively. Phagocytosis can be measured as a function of the percentage of CD11b+CFSE+ cells, representing macrophages that have bound/ingested target cells. A. Co-culture of macrophages with the 697 cells in the presence of mIgG1 (left plot) resulted in a minimal basal level of phagocytosis. Blocking with anti-CD47 (right plot) increased phagocytosis tenfold. B, C. The basal levels of HepG2 and Caco-2 cell phagocytosis were higher when compared to the 697 cells. Phagocytosis was not appreciably changed by blocking with anti-CD47, probably due to the low levels of CD47 expression in these cell lines.

The results are shown in Figure 2. Blocking CD47 in the 697 cells (Figure 2A) resulted in a tenfold increase in phagocytosis (10.3% vs 1.0%), confirming that CD47 plays a role in the inhibition of phagocytosis in this cancer cell line. HepG2 and Caco-2 cells expressed lower levels of CD47 as compared to the 697 cells and blocking with anti-CD47 did not affect phagocytosis of these cells (Figures 2B and 2C).

This experiment demonstrates how flow cytometry assays of four or fewer colors on the BD Accuri C6 Plus can assess the expression levels of target antigens involved in inhibition of immune function as well as measure phagocytic activity of macrophages.

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


 

Tips & Tricks

Attenuation filters

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Figure 3. Using an attenuation filter to bring bright signals on scale
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Figure 3.Using an attenuation filter to bring bright signals on scale

PBMCs were frozen and stored at –80°C for approximately two weeks, resulting in a mixed live/dead population. Cells were thawed and stained with BD Calcein AM (Cat. No. 564061) at concentrations of 10 μM, 5 μM or 2.5 μM in serum-free buffer. Data was acquired on a BD Accuri C6 flow cytometer with or without the use of a BD Accuri™ FL1 90% Attenuation Filter and analyzed using BD Accuri C6 software. Cells were initially gated based on light-scatter properties to identify lymphocytes (not shown). Results: Live cells were identified using their Calcein AM fluorescence profiles. The 10-μM titration resulted in the best resolution of live vs dead cells (left plots), but the unattenuated signal (A) was off-scale. Further dilution of the dye would bring the signal on scale but would compromise the resolution of live vs dead cells. Thus, signal attenuation (B) was necessary to visualize calcein fluorescence while maintaining optimal resolution.

The BD Accuri C6 Plus system can detect and analyze fluorescence signals across an exceptional seven-decade dynamic range, from dim to bright. In rare cases, however, signals of extreme brightness can appear off-scale. Examples include signals from fluorescent proteins (depending on transfection efficiency, the level of expression within the cells and the excitation wavelength used), Calcein AM and CFSE, as discussed in this month’s Application Highlight.

In such cases, an easily inserted attenuation filter can bring the signals back on scale, further increasing the usable range of the instrument. This is advantageous because the detectors used to read these bright signals will still be operating within their linear range. Attenuation filters for BD Accuri™ cytometers are available at 90% and 99% efficiency for each fluorescence detector, reducing brightness by one or two magnitudes.

Figure 3 shows peripheral blood mononuclear cells (PBMCs) stained with the viability dye Calcein AM at three levels of titration. The 10-μM titer achieved the best resolution, but the fluorescence was off-scale. The BD Accuri™ FL1 90% Attenuation Filter (Cat. No. 653173) was used to reduce brightness by an order of magnitude, bringing the calcein fluorescence back on scale. The BD Accuri™ FL1 99% Attenuation Filter (Cat. No. 653172) would reduce brightness by an additional order of magnitude.


 

Publication Picks

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

Nanoparticle-mediated gene therapy

Basoglu H, Goncu B, Akbas F. Magnetic nanoparticle-mediated gene therapy to induce Fas apoptosis pathway in breast cancer. Cancer Gene Ther. 2018; doi: 10.1038/s41417-018-0017-2. PubMed

Nanogel-based antiviral vaccine

Nuhn L, Van Hoecke L, Deswarte K, et al. Potent anti-viral vaccine adjuvant based on pH-degradable nanogels with covalently linked small molecule imidazoquinoline TLR7/8 agonist. Biomaterials.2018; doi: 10.1016/j.biomaterials.2018.03.026. PubMed

Designer epigenome modifiers

Mlambo T, Romito M, Cornu TI, Mussolino C. Delivery of designer epigenome modifiers into primary human T cells. Methods Mol Biol. 2018;1767:189-203. PubMed

Aquaculture-wastewater plume

Hozumi A, Hong PY, Kaartvedt S, Røstad A, Jones BH. Water quality, seasonality, and trajectory of an aquaculture-wastewater plume in the Red Sea. Aquacult Environ Interact. 2018;10:61-77. Abstract


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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