BD ACCURI NEWS
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.
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.
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.
Tips & Tricks
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.
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
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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