Countess Automated Cell Counter Applications

Using the Countess 3 FL Automated Cell Counter, cell viability can easily be determined with colorimetric or fluorescent stains. Both the colorimetric SafeCount Viability Stain and the fluorescent ReadyCount Green/Red Viability Stain provide fast, accurate, and reproducible measurements with minimal hands-on time. SafeCount stain is a brightfield, safer alternative to Trypan Blue for use on Countess 3 systems, reducing precipitation to improve consistency and reliability in viability measurements (Figures 1 and 2).
 

ReadyCount stains enable fluorescence-based discrimination of live and dead nucleated cells in under five minutes. By using fluorescent signals to identify cells, fluorescence counting improves accuracy by excluding debris and non-cellular particles visible in the brightfield image. In addition, fluorescent counting can use a dedicated algorithm distinct from the brightfield algorithm, enabling more sensitive detection and analysis of specifically stained cells. The EVOS GFP 2.0 light cube quantifies the live cells and the EVOS Texas Red 2.0 light cube quantifies the dead cells, establishing accurate viability results (Figure 1B). 

Fluorescent staining is a powerful tool for microscopy, flow cytometry, and cell counting, enabling selective detection of targets while minimizing interference from debris and supporting multiplexed analyses. ReadyCount stains are ready-to-use, benchtop-stable fluorescent nuclear stains optimized for the Countess 3 FL Automated Cell Counter. They enable fast, accurate enumeration of nucleated cells, even in complex samples containing mixed cell populations or cellular debris.

ReadyCount Blue Nuclear Stain uses Hoechst dye to label all nucleated cells (Figure 3A), providing clear fluorescent identification for reliable cell counting. Because Hoechst staining intensity can vary across cell types—particularly hematopoietic cells—optimization of staining time may be required to achieve consistent results. Countess 3 FL software further enhances analysis by enabling visualization of single-cell fluorescence intensity through on-screen scatter plots (Figure 3B), supporting deeper insights into sample composition.

There can be numerous challenges when assessing cell health in freshly harvested peripheral blood mononuclear cells (PBMCs, also called white blood cells or WBCs). Blood is a rather complex sample type, as it contains various components that can lead to difficulty in manual cell counting. Countess 3 automated cell counters can significantly improve the accuracy of results while saving time over manual cell counting.

Human and murine PBMCs are notoriously challenging to count due to their small size and low contrast. The manual hemocytometer is the most commonly used instrument for determining cell concentrations. However, the task is tedious, requires careful cleaning and handling of the hemocytometer, and is subject to variation between users. Less variation and more reliable measurements are seen in both human and murine PBMCs when using the Countess 3 FL Automated Cell Counter compared to manually counting with a hemocytometer (Figures 4 and 5). 

Single-cell RNA sequencing (scRNA-Seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-Seq) technologies enable new insights into gene expression and regulation by providing data resolution at the single-cell level. To obtain high-quality single-cell data, protocols for scRNA-Seq and scATAC-Seq require an accurate count of viable suspended single cells or nuclei as input, with minimal presence of cellular aggregates and dead cells. The Countess 3 FL Automated Cell Counter is designed for accurately counting cells and nuclei, evaluating their viability, and assessing aggregation (Figure 6). 

The ability to assess cell health in a population is a basic yet critical evaluation parameter in many cell and molecular biology labs. What used to take more than an hour on traditional microscopes or cytometers—simply acquiring preliminary results—takes only a few minutes with the Countess 3 FL Automated Cell Counter. Save time and effort by checking your sample before using a microscope or flow cytometer.

Immuno-oncology research continues to grow as therapies such as CAR-T cells, checkpoint inhibitors, and T cell–engaging bispecific antibodies improve the ability of T cells to target and kill cancer cells. During T cell expansion and immunotherapy development, the Countess 3 FL Automated Cell Counter delivers fast, accurate measurements of cell viability and concentration before and after isolation and activation, helping preserve valuable cells and streamline workflows. In the example below, CAR-T cells were isolated using the Dynabeads Untouched Human T cells Kit and activated using the Dynabeads Human T-activator CD3/CD28 for T Cell Expansion and Activation where cell culture was monitored before and after expansion (Figure 9).

With the Countess 3 FL Automated Cell Counter, you can quickly verify what proportion of your cells have been transfected or transduced successfully before moving on to downstream experiments and analysis. Transduction efficiencies are critical to CRISPR-Cas9 systems as well as in the expression of a fluorescence protein reporter, such as GFP. In genome editing experiments using CRISPR-Cas9, lentiviral transduction is often used to help deliver the CRISPR-Cas9 complex efficiently into the cells. Control particles that express a fluorescent protein (such as GFP) and potentially knock out other genes (such as HPRT), are frequently used to measure transduction efficiency. The Countess 3 FL Automated Cell Counter can be used to quantitate both viability and transduction efficiency in these cells (Figure 10).

Oxidative stress and mitochondrial function are key indicators of cellular health, impacting cell viability, metabolism, and overall experimental outcomes. Fluorescent assays targeting reactive oxygen species (ROS) production and mitochondrial activity provide valuable insights into cellular responses to stress, drug treatment, and disease states.

The Countess 3 FL Automated Cell Counter enables rapid quantification of these parameters through fluorescence-based detection, allowing researchers to assess cell health while simultaneously measuring cell concentration and viability. Combined with intuitive analysis software and multi-channel fluorescence capabilities, the Countess 3 FL instrument streamlines workflows by delivering fast, reproducible results without the complexity of traditional flow cytometry, making it an excellent solution for routine monitoring of cell cultures and functional cellular assays.

Flow cytometry is a powerful method for characterizing complex cell populations, delivering rich information on phenotype, viability, and function. But for many routine workflows, researchers first need a faster, simpler way to evaluate cell concentration, viability, and fluorescence-based markers before committing samples to more comprehensive analyses.
 

Used as a preliminary step before flow cytometry or fluorescence-assisted cell sorting (FACS), the Countess 3 and Countess 3 FL Automated Cell Counters can help improve experimental success while conserving valuable time, money, and sample. Figure 12 demonstrates strong comparability in percent positive across three fluorescent viability assays, underscoring the utility of Countess data as an accurate and reliable front-end (or replacement) screen for downstream workflows. The Countess 3 FL instrument enables users to detect fluorescent cell emission across the spectrum, from violet (DAPI) to NIR (Cy7), with simultaneous observation of up to two fluorophores.