Once upon a time, flow cytometers were big, bulky instruments most often run by highly skilled operators in core facilities. With the advent of compact, user-friendly personal flow cytometers, many functions and capabilities of advanced multicolor, multiparametric flow cytometry are moving to the individual laboratory. Instruments such as the peristaltic pump-driven BD Accuri™ C6 Plus are equally at home on a cancer research or molecular biology bench as in a mobile limnology lab, performing tasks such as immunophenotyping and functional assays, gene expression studies, queries of activation, and even concentration measurements and absolute cell counting.
Figure 1. Analyzing a cell by flow cytometry
After cells are labeled with fluorescently conjugated antibodies or dyes they pass through the flow cytometer, are excited by lasers, and emit light proportional to the amount of fluorophore excited by that laser. Different fluors can be excited by the same wavelength yet emit at different wavelengths, and so the number of “colors” that can be discriminated—determined by the instrument’s optical filters and detectors—will often exceed the number of lasers. The Accuri C6 Plus, for example, boasts two lasers and four “color” channels, plus two scatter detectors, for a total of six parameters. And since thousands of particles are typically interrogated from a given sample, robust statistics can be generated relating those parameters. This article samples some of the life science applications to which personal flow cytometers such as the Accuri C6 Plus are being put to use.
Flow cytometry is the ideal tool to determine the distribution of cells in a population. For example, it’s straightforward to determine the lymphocyte composition of a blood sample. Using antibodies against CD3 and CD19 will distinguish T cells (CD3+, CD19-) from B cells (CD3-, CD19+) from other cell types (CD3-, CD19-). Additional antibodies and dyes can be used to simultaneously determine other parameters such as activation state.
Say you want to know whether the T cells are CD4+ or CD8+, and whether the B cells express the Igλ or IgΚ light chain. If your instrument has only four fluorescent channels, you would think you’d have to run two separate experiments—one staining for CD3, CD19, CD4, and CD8, and another staining for CD3, CD19, Igλ, and IgΚ. But because you know that cells won’t be both CD3+ and CD19+ (you’ve verified this, just to be sure), you can use the same channel for CD4 and Igλ, and the same channel for CD8 and IgΚ. Then, by gating on CD3+ cells you can plot CD4+ vs. CD8+ T cells. And by gating on CD19+ cells you can plot Igλ+ vs. IgΚ+ B cells. All in a single experiment, saving time, effort, sample, and reagents.
Figure 2. Sentinel analysis of T and B cells Lysed whole blood was stained with two unique fluorochromes for the sentinel markers CD3 (PerCPCy5.5 and CD19 (APC), defining T and B sentinel cell types respectively. Two other fluorochromes (FITC and PE) were used as described for markers that are unique to one sentinel cell type: CD4 and CD8 within T cells and immunoglobulin lambda and kappa light chains (Ig lambda and Ig kappa) within B cells. A. Cells were stained with CD3 PerCP-Cy5.5, CD19 APC, CD4 PE, and CD8 FITC. Analysis confirmed that CD4 and CD8 were expressed exclusively by T cells (gated on CD3+, middle plot) and not by B cells (gated on CD19+, right plot). B. Similarly, when cells were stained with CD3 PerCP-Cy5.5, CD19 APC, Ig lambda FITC, and Ig kappa PE, only CD19+ B cells expressed Ig lambda and kappa. These panels (A and B) confirmed that CD4/ CD8 and Ig lambda/kappa were uniquely expressed in two sentinel cell types, T and B cells respectively, and the same fluorochromes could then be used doubly for these markers. C. Cells were stained with CD3 PerCP-Cy5.5, CD19 APC, CD4 PE, Ig kappa PE, CD8 FITC, and Ig lambda FITC. Both T-cell subsets (CD4+ and CD8+, middle) and B-cell subsets (Ig lambda+and Ig kappa+, right) were clearly distinguished in a single panel using four fluorochromes paired with six markers.
In addition to cell surface markers, flow cytometry can query intracellular molecules as well. The fluorochromes may be genetically expressed, like green fluorescent protein (GFP) and its kin, and serve as reporters. Fluors conjugated to antibodies may be introduced to permeabilized cells to identify endogenous intracellular proteins such as the stem cell pluripotency marker Oct3/4, or proteins that are destined to be secreted. Fluorescent dyes, too, can be used to look inside the cell. For example, cell-permeable dyes are commonly used to stain nuclear material for querying cell cycle phase and ploidy, while cell-impermeable dyes—which enter the cell through damaged membranes—are an easy way to assess viability.
And by using a multiplex assay such as the BD™ Cytometric Bead Array (CBA) method, secreted proteins can be captured from bodily fluids or culture supernatant and interrogated by flow cytometry. Combining intracellular staining with CBA allows for a more comprehensive understanding of secretion kinetics.
Researchers often want to know not only if a cell is dead (or dying), but how and when the cell dies—say, to better assess the effects of a perturbation. Unlike healthy cells, early apoptotic cells display phosphatidylserine (PS) on the cell membrane’s outer leaf, and this is commonly detected using labeled Annexin V. Other events characteristic of different stages of apoptosis, such as mitochondrial membrane depolarization, caspase activation, and DNA fragmentation, can simultaneously be detected by flow cytometry to give a more comprehensive measure of the impact of a drug’s kinetics and mechanisms of action on the single cell, for example.
Figure 3. Time course of apoptosis in Jurkat cellsJurkat (ATCC® TIB-52) cells were treated with 100 ng/mL of FAS ligand (Cat. No. 556375) to induce apoptosis, or with FAS ligand and BD Pharmingen™ Q-VD-OPh General Caspase Inhibitor (20 μM), or were left untreated. Cell viability was analyzed at 3, 6, and 24 hours. Cells were stained with BD Pharmingen™ Propidium Iodide Staining Solution (Cat. No. 556463) and BD Pharmingen™ APC Annexin V (Cat. No. 550474) and analyzed on the BD Accuri C6 Plus. Results: Over time, cells treated with FAS ligand (middle column) progressed from live to apoptotic to dead. The majority of cells treated with both FAS ligand and Q-VD-OPh (right column) remained viable, comparable to untreated cells (left column).
Even when cells are viable, they may be quiescent or they may be cycling. There are a variety of ways that flow cytometry can be used to look at cell proliferation, from examining the incorporation of a dNTP analog such as BrdU or EdU (using click chemistry), to staining for markers such as Ki-67, to dyeing the cells with CFSE, which results in the fluorescent intensity of the cell halving with each new generation.
These and other cellular processes are tightly regulated through protein phosphorylation signaling cascades. Flow cytometry allows multiple key phospho-sites to be quantitatively examined on a per-cell basis, in conjunction with other markers, providing a level of information not obtainable by more traditional assays such as Western blotting.
Calcium signaling also can be assayed by flow cytometry with dyes that indicate the Ca2+ flux (from intracellular stores, for example). Ca2+ levels can change very rapidly upon stimulation. A flow cytometer like the Accuri C6 Plus that utilizes a nonpressurized, open fluidics system allows test compounds to be added on the fly, enabling nonstop monitoring and accurate, gap-free dynamic Ca2+ measurement of each cell in the population.
By using a fluorescent reporter, gene knock-outs, knock-ins, and knock-downs (such as by siRNA and miRNA), as well as the effect of other perturbations to the cell, can be monitored by flow cytometry.
Flow cytometry can be used to monitor transfection efficiency. Comparing different conditions (for example, the amount of plasmid and reagent) allows for a quick and easy way to optimize a transfection protocol. Flow cytometry can detect the gradient of GFP expression in a large number of cells of distinct phenotypes, thereby identifying the subsets of cells with a given copy number of the expression vector.
Similarly, the efficiency of knockout by CRISPR/Cas9 technology can be quantified using a fluorescently tagged insert.
Flow cytometry isn’t just for mammalian cells. An instrument like the Accuri C6 Plus with an adjustable core diameter and broad dynamic range can obtain data from microbes of varying size and fluorescence. Light scatter can reveal some basic information about size, shape, and surface features of bacteria and yeast, for example. Fluorescent dyes can be used—like with mammalian cells—to assess viability, metabolic activity, and concentration. In some instances, natural autofluorescence can be used to discriminate between types of organism, for example distinguishing cyanobacteria from red algae in several water sources based on phycoerythrin and phycocyanin fluorescence.
Figure 4. Algae analysis on the BD Accuri C6 PlusIn three environmental water samples, autotrophic phytoplankton were identified based on detection of chlorophyll a and b, and characterized as blue-green algae (cyanobacteria) or red algae based on Phycoerythrin and Phycocyanin fluorescence. Discrimination of phytoplankton from background noise was achieved by triggering on FL3 and by setting an appropriate threshold value. Coastal and bay water contained a variety of algae with distinct autofluorescence signatures, while chlorinated pond water contained a single dominant population.
Small, rugged, transportable flow cytometers such as the Accuri C6 Plus can also be taken on the road for environmental studies. Its optics and fluidics, for example, allow for continuous operation even during motion and vibration.
Flow cytometry can complement, and sometimes replace, techniques such as qPCR, fluorescent microscopy, ELISA, and Western blotting, quickly yielding statistically robust, multiparametric data on large collections of individual cells. With smaller, easier to use instruments, prepared kits, and downloadable software templates, flow cytometry has broken the tether of the core facility, democratizing its use in more and more life science applications.