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Knowing the cell as well as their populational distribution in complex cellular networks is of uttermost importance when analyzing cell cultures, blood or disseminated tissue for understanding disease processes and the underlying biology. Revealing cellular functions by cytometric scrutinizing of surface antigen expression patterns is possible by surface antigen staining. This was demonstrated by the OMIP-015 by Mahnke and coworkers (this issue, page 179) who characterized human regulatory and activated T-lymphocytes in the absence of intracellular cytokine staining. Still, avoiding the direct functional analysis of intracellular pathways or secretion activities of individual cells has its limitations. As an example, different modes of programmed cell death (apoptosis, programmed necrosis or autophagy) (1) that are of relevance in tumor development and actions of antitumor drugs need the deeper investigation of intracellular signaling pathways.

Traditional intracellular cytokine assays by flow cytometry can precisely determine which cell type (characterized by a specific surface antigen phenotype pattern) is able to produce which pattern of cytokines or chemokines. An excellent actual example is the OMIP-016 by Guenounou and coworkers (this issue, page 182). This group presents a 10-color flow cytometry assay for the characterization of primate T-cells' antigen response. Even so, cytokines, chemokines, and other agents make their biological action as communicators with other cells, similar to neurons, by being excreted. Unfortunately, measuring intracellular production of secreted peptides and proteins requires the manipulation of intracellular pathways by blocking Golgi and endoplasmatic reticulum secretion with agents such as Brefeldin-A combined with the opening of the cell membrane with detergents to allow detecting antibodies into the cells.

In the past, revolutionary single cell methods for flow cytometry have been introduced to fill this gap and to detect in less perturbed individual cells which substances they release as a response to a specific stimulus. As commented by Trautmann (this issue, page 177), initially flow cytometry was developed to measure cell-bound constituents, may they be on or within the cell. Revolutionary work with regard to ligand secretion by single cells was done by embedding single cells into a matrix and staining and subsequently measuring secreted peptides therein (2). Alternatively, bivalent antibodies were used that on the one hand tagged the specific cell of interest and on the other hand captured secreted peptides. Those in turn were labeled by fluorochrome tagged sandwich antibodies (3). Although both approaches are very elegant, they require substantial technical expertise to be performed at reasonable quality and thus are not simple to handle for many standard laboratories.

Now, Fitzgerald and Grivel (this issue, page 205) made a major step forward by developing a novel nanoparticle-based single cell secretion capture assay that is universally applicable and fairly simple to use. This method can be used for detection of secretion in live cells and thereby allows cell sorting methods for further enrichment and culturing. The authors used magnetic nanoparticles that were labeled with antibodies against mouse antibodies and purified them using a magnet. Then the peptides were, on the one hand, tagged with a targeting antibody that would dock onto the cells of interest and on the other hand with a capturing antibody for the secreted substance. When this substance is secreted, it binds to the capturing antibody and is identified using a fluorochrome labeled sandwich antibody. This assay is universal as the nanoparticles can be tagged with any targeting and capturing antibody of interest. In addition, differently tagged nanoparticles may be combined in a single assay in order to detect multiple ligand secretion on the single cell level. Because of its relative simplicity, this novel capturing assay may increase our knowledge on cytokine secretion under near biological conditions.

Another important question is how to monitor in viable cells the trafficking of proteins—naturally, for us as cytometrists, on the single cell level! Wu and coworkers (this issue, page 220) used their fluorogen-activating protein (FAP) technology (4) and high-throughput flow cytometry to study multiple trafficking patterns of G-protein coupled receptors, simultaneously. The authors emphasize that the cross talk of different types of these receptors are relevant for several economically important diseases in the world, including cardiac diseases and cancer. Using FAP, fluorescence signals can be enhanced as much as by a factor of 10,000 and thus are substantially brighter than fluorescent proteins (5). The authors demonstrate that by combining different FAPs enhancing different fluorescent dyes emitting at different wavelengths is possible and the interaction of two different receptors in the same cell can be quantified. Similar to the coexpression of four or more fluorescent proteins in the same cell (6) highly multiplexed FAP experiments can be envisioned. Taking the high sensitivity of this method into account, multiplexed FAP assays will provide deeper insight into complex heterogeneity of cellular transduction and transport patterns in the individual cell.

And Now for Cell Proliferation and Death …

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  2. And Now for Cell Proliferation and Death …
  3. Literature Cited

Viability assays by cytometry technologies are of substantial relevance in as diverse fields of cell research as drug discovery and screening, basic research of cell death processes and drug testing of patients' tumor cells for individualized therapy. For this task, image and flow cytometry are applied and a set of dyes for probing death and viability are available. Still, there is a need for improved dyes as well as reliable assay evaluations.

The international group of Akagi and coworkers (this issue, page 227) developed a novel far red emitting viability dye DRAQ7. This dye is a further development of the well-known viable DNA label from the anthracycline family, DRAQ5, that was extensively used in flow and image cytometry (7 ). In contrast to DRAQ5, DRAQ7 is only entering cells with a compromised cell membrane, thus indicating onset and progression of cell death. The dye has a low cytotoxicity and can thereby be used for long-term (>24 h) real-time monitoring in cytotoxicity experiments by flow or image cytometry. DRAQ7 can be tested also in combination with other viability dyes, like the mitochondrial membrane potential sensitive dye tetramethylrhodamine methyl ester, among others. The authors emphasize that DRAQ7 provides the foundation for novel automated and high-throughput screening assays for drug discovery and drug testing in individualized therapy and also in fragile cells.

The comparability of cell viability assays measured by flow or by image cytometry was critically evaluated by Kummrow and coworkers (this issue, page 197). Using isolated CD14+ monocytes or CD34+ stem cells from the peripheral blood of healthy humans as a test system and live/dead staining with calcein acetoxy-methyl-ester/ethidium homodimer-1, the authors compared under highly standardized conditions results obtained by both cytometry technologies. In conclusion, image and flow cytometry yield nearly identical results for both the fraction of dead and live cells.

Literature Cited

  1. Top of page
  2. And Now for Cell Proliferation and Death …
  3. Literature Cited
  • 1
    Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, Bao JK. Programmed cell death pathways in cancer: A review of apoptosis, autophagy and programmed necrosis. Cell Prolif 2012; 45: 487498.
  • 2
    Manz R, Assenmacher M, Pflüger E, Miltenyi S, Radbruch A. Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix. Proc Natl Acad Sci USA 1995; 92: 19211925.
  • 3
    Brosterhus H, Brings S, Leyendeckers H, Manz RA, Miltenyi S, Radbruch A, Assenmacher M, Schmitz J. Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion. Eur J Immunol 1999; 29: 40534059.
  • 4
    Wu Y, Tapia PH, Fisher GW, Simons PC, Strouse JJ, Foutz T, Waggoner AS, Jarvik J, Sklar LA. Discovery of regulators of receptor internalization with high-throughput flow cytometry. Mol Pharmacol 2012; 82: 645657.
  • 5
    Szent-Gyorgyi C, Schmidt BF, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JA, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat Biotechnol 2008; 26: 235240.
  • 6
    Tiemann U, Sgodda M, Warlich E, Ballmaier M, Schöler HR, Schambach A, Cantz T. Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells. Cytometry A 2011; 79A: 426435.
  • 7
    Grimwade L, Gudgin E, Bloxham D, Bottley G, Vassiliou G, Huntly B, Scott MA, Erber WN. Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry. Cytometry A 2012; 81A: 896900.