Fluorescence-Based Viability Assays for the Countess II FL Automated Cell Counter

Incorporate automation into your cell analysis workflows

(See a list of the products featured in this article.)

Many biological research applications require accurate and precise measurement of cell number and viability prior to downstream analysis. The Countess™ II FL Automated Cell Counter is a three-channel (brightfield and two optional fluorescence channels) benchtop assay platform with state-of-the-art optics and image analysis software for automated cell counting. With autofocusing and auto–brightfield lighting controls, as well as multiple gating settings (size, brightness, circularity, fluorescence intensity), this automated counter allows researchers to analyze cells in suspension for a range of characteristics, including cell viability, cell cycle phase, apoptotic stage, and fluorescent protein expression. A number of Molecular Probes™ fluorescence-based assays are compatible with the Countess II FL instrument, which enables quick and easy checks of the health of your cells before committing them to existing workflows for imaging, flow cytometry, and high-content analysis (HCA).

Minimize counting variability

When manually counting cells using a hemocytometer and a microscope, count-to-count variability of a single sample by an experienced cell biologist is typically 10% or more. Counting variability between multiple users commonly exceeds 20%. In addition to helping save time, automated cell counters minimize the subjective nature of manual counting as well as user-to-user differences in total cell count assays. Users of the Countess II FL Automated Cell Counter typically observe count-to-count variabilities of less than 5%, while spending as little as 10 seconds per sample count.

Assessing cell viability is both a key step in daily cell manipulation and often a requirement for subsequent processing and analysis. Moreover, knowing the viability of your cell sample prior to application of costly reagents or booking core facility time can be extremely valuable in today’s fast-paced research environment. Trypan blue staining coupled with brightfield imaging is a common method for viability analysis, but many fluorescence options are becoming commonplace in both microscopy and flow cytometry workflows. With fluorescence-based viability assays on the Countess II FL Automated Cell Counter, you can simplify your workflow by obtaining cell count and viability information directly from your samples, immediately prior to advanced analysis.

Detect cell viability with a one-color fluorescence assay

There are many choices for one-color fluorescence viability assays, depending on your experimental needs. For assays where you need to distinguish live cells from dead cells after a fixation step, we recommend the LIVE/DEAD™ Fixable Dead Cell Stains. Only requiring a single fluorescence channel, the LIVE/DEAD Fixable Dead Cell Stains enable discrimination of live and dead cells based on membrane permeability and amine-reactive fluorescent dye chemistry. In viable cells, only surface proteins are available to react with these probes, resulting in relatively dim staining; conversely, in dead cells with compromised plasma membranes, these probes react with both intracellular proteins and those on the cell surface to produce intense fluorescent staining. The roughly 50-fold difference in intensity between live and dead cells is easily distinguished on the Countess II FL instrument equipped with standard EVOS™ Light Cubes (Figure 1). Figure 1B shows that the results of dead-cell detection with the LIVE/DEAD Fixable Dead Cell Stains are comparable with those obtained using the conventional trypan blue assay.

When a fixable dead-cell indicator is not required, we offer several other single-color viability markers—including the cell-impermeant propidium iodide, ethidium homodimer, and SYTOX™ nucleic acid stains—which selectively stain dead cells that have compromised plasma membranes. These nonfixable nucleic acid stains are commonly employed for multiplex analysis with functional or structural fluorescent probes, including fluorescent proteins or cell-surface markers. Figure 2 demonstrates the ability of the Countess II FL instrument equipped with a Cy®5 EVOS Light Cube to count dead cells stained with the SYTOX Red Dead Cell Stain. A total cell count can be acquired using the brightfield image, providing a simple method for calculating the ratio of live (unstained) to dead (SYTOX Red stained) cells within the population (Figure 3).

Figure 1. Cell viability detection using single-color viability assays. Live and ethanol-killed Jurkat cells were mixed (~2:1 live:dead), stained with trypan blue or with various colors of the LIVE/DEAD™ Fixable Dead Cell Stains, and analyzed on the Countess™ II FL Automated Cell Counter equipped with the appropriate EVOS™ Light Cubes. (A) A cell population treated with LIVE/DEAD Fixable Green Dead Cell Stain for 30 min at 37°C is analyzed with the Countess II FL instrument equipped with the GFP EVOS Light Cube. Dead cells represented 41% of the total cell population as seen by positive staining. (B) For each stain, a total cell count was acquired using the brightfield image. A dead cell count for trypan blue staining was determined by brightfield (BL) or fluorescence (FL) imaging. For the LIVE/DEAD Fixable stains, the dead cell count was determined using the fluorescence channel. The percentages of dead cells indicated by all the dyes are in good agreement, but differ from the theoretical 2:1 starting ratio (33%) because the “live” population contained some dead cells.

Figure 2. Discrimination of live and dead cells on the Countess II FL Automated Cell Counter. Live and formaldehyde-killed Jurkat cells were mixed (~1:1), stained with SYTOX™ Red stain, and analyzed on the Countess™ II FL instrument equipped with the Cy®5 EVOS™ Light Cube. A total cell count was acquired using the brightfield image, and a SYTOX Red–positive cell number was generated using the fluorescence channel, allowing the percentage of dead cells to be calculated for the population.

Figure 3. Cell viability assay with SYTOX Red Dead Cell Stain. Live and formaldehyde-killed Jurkat cells were mixed in the indicated ratios, stained with SYTOX™ Red stain, and analyzed using the Countess™ II FL instrument equipped with the Cy®5 EVOS™ Light Cube (see example in Figure 2).

Detect cell viability with a two-color fluorescence assay

A more robust determination of cell viability can be achieved using an assay that simultaneously measures two different parameters of cell health. We recommend using the LIVE/DEAD™ Viability/Cytotoxicity Kit with the Countess II FL Automated Cell Counter. This viability assay relies on the use of two fluorescent probes: ethidium homodimer-1 is a high-affinity, red-fluorescent nucleic acid stain that is only able to pass through the compromised membranes of dead cells, and calcein AM is a cell-permeant fluorogenic esterase substrate that is hydrolyzed by active esterases to a green-fluorescent product (calcein) upon entering a live cell. Cells stained using the LIVE/DEAD Viability/Cytotoxicity Kit can be evaluated on the Countess II FL instrument equipped with Texas Red™ and GFP EVOS Light Cubes (Figure 4). With the viability assessment in hand, the cell sample can then be subjected to further functional analysis on other imaging platforms or by flow cytometry.

Figure 4. Cell viability assay with the LIVE/DEAD Viability/Cytotoxicity Kit. Live and heat-killed cells were mixed, stained with calcein AM and ethidium homodimer-1 supplied in the LIVE/DEAD™ Viability/ Cytotoxicity Kit, and analyzed on the Countess™ II FL instrument equipped with GFP and Texas Red™ EVOS™ Light Cubes. The histogram shows the counts of live cells in the population, which fluoresce green, and of dead cells, which fluoresce red.