Subcellular organelles

Flow cytometry is a well-established, high-throughput single-particle analysis and purification technique with broad applications in biomedical and environmental science. The two requirements for flow cytometry samples are that the particles (cells, beads, organelles, etc.) are in a disaggregated suspension and are small enough to fit through the fluidics system without disrupting sheath flow. Some biomedical specimens such as blood, bone marrow, spleen, and cell lines are easy to prepare and generate well-defined patterns in the resulting data. Other sample types are much more challenging. Solid tissues and tumors must undergo extensive mechanical and enzymatic digestion to release individual cells, snap-frozen tissues require both disaggregation and nuclear isolation. These harsh processing steps not only release live cells, but also generate large amounts of debris and dead cells that complicate flow cytometric analysis and sorting. Staff members in flow cytometry shared resource laboratories need to have options for approaching challenging samples to ensure the highest chances of success for the researchers. Here, I will present three strategies to help identify target particles in challenging samples.

Mix it up! There is more than one way to gate a population. The default first plot for flow cytometry is forward scatter vs. side scatter. These parameters allow the cytometrist to evaluate morphological properties of the sample quickly and identify cellular patterns. In samples containing large amounts of debris and dead cells, data patterns may be obscured. To reduce overwhelming background noise, try gating for live cells first by looking at a plot of forward scatter vs. viability dye, or viability dye vs. side scatter. This plot will show dead cells as viability-dye-bright events, and non-nuclear debris will form a waterfall pattern back to the axis revealing the live cell population for gating (Figure 1.) The gated live cells may then be assessed for other markers, aggregation status, and morphology.

Two dyes are better than one! Use a combination of cell-permeable and cell-impermeant dyes. In some cases, changing the gating strategy as discussed above may not be sufficient to identify low numbers of live cells in a highly degraded sample. Measurements of scatter are less sensitive than fluorescence. We can take advantage of the different properties of DNA-binding dyes to make our live cells fluoresce in a unique, identifiable color. Popular viability dyes such as DAPI and propidium iodide rely on an intact plasma membrane to exclude them from entering the cell and gaining access to its DNA. Recent advances in fluorochrome technology have brought cell-permeable DNA dyes to the market to address the need for measuring DNA content in live cells. We can pair an older membrane-excluded dye such as DAPI with a new live cell DNA dye such as Vybrant® DyeCycle™ Ruby to generate a sample where dead cells are dual labeled and live cells fluoresce brightly in only the Vybrant® DyeCycle™ Ruby channel allowing us to differentiate them from background (Figure 2.) We can then assess our live cell population for other characteristics.

Use your landmarks! Isolated nuclei should display a distinct cell cycle pattern. Sometimes researchers work with snap-frozen tissue from biobanks or collaborating institutions. The freezing process destroys cellular integrity, but the nuclei containing valuable genomic information are often intact and locked in their tissue-resident state. Isolation of these nuclei enables researchers to access information about diseases and tissues that would otherwise be inaccessible.

Nuclei are smaller than most of the cells that come into a biomedical flow lab and can be difficult to identify by scatter properties. To improve detection of nuclei, we again turn to DNA-binding dyes. Because the nuclei no longer receive protection from a plasma membrane, we can incubate them with dyes such as DAPI or propidium iodide so that they are strongly fluorescent. In addition to intact nuclei from healthy cells, these samples may contain other nucleic-acid-containing particles such as mitochondria and apoptotic nuclei. To distinguish healthy nuclei from this background we measure DNA content. DNA dyes bind stoichiometrically and, under saturating dye conditions, flow cytometry can distinguish the differences in DNA content based on the relative brightness of fluorescence in the DNA-binding dye channel. Cells in the G1/G0 phase of the cell cycle have 2N chromosomes, cells in G2/M have 4N chromosomes and appear twice as bright as the 2N cells on a linear scale. Apoptotic nuclei, nuclear fragments, and mitochondria will all have sub-G1/G0 fluorescence intensity and can be excluded from analysis or sorting (Figure 3.)