Single-cell genomics applications such as single-cell RNA sequencing (scRNA-seq) and Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) are widely used for characterizing individual cells within a complex population. However, to achieve reliable results, researchers must isolate nuclei from sample material and accurately distinguish them from intact cells and debris. Here, we explain why Acridine Orange/Propidium Iodide (AO/PI) staining is preferred over Trypan Blue staining for monitoring total cell number and viability and counting isolated nuclei. We also comment on how using an automated cell counter to perform AO/PI measurements can benefit single-cell genomics research.
Unlike conventional bulk population sequencing methods, single-cell genomics techniques highlight cell-to-cell variability for deeper insights into sample material. For example, scRNA-seq allows researchers to compare cellular transcription profiles between conditions of health and disease, while ATAC-seq provides information about chromatin accessibility and its impact on gene expression. Yet, the value of such studies depends on accurate counting of isolated nuclei, since library preparation requires that the number of unlyzed cells is minimized and samples are free of cellular aggregates and debris. Additionally, many single-cell genomics techniques demand intact nuclei to function correctly.
Isolated nuclei are typically counted using cell viability applications. A common approach is to use Trypan Blue, a dye that is excluded from intact cells to leave the nuclei stain-free. However, because Trypan Blue staining is not always able to accurately resolve nucleated cells from debris, it often underestimates the total cell number and overestimates the viability. For this reason, AO/PI staining is preferred—both for routine cell viability testing and for single-cell genomics studies.
Figure 1. Automated image analysis of trypan blue stained cells
During cell viability testing, the AO/PI fluorophore combination stains live cells so they fluoresce green and dead cells so they fluoresce red. Critically, it avoids the detection of cell debris. The methodology has more recently been adapted for single-cell genomics applications, where the green and red staining corresponds to intact cells and successfully isolated nuclei, respectively. This strategy enables researchers to simultaneously calculate residual unlyzed cells as a percent of the total cell number, and count nuclei, providing an indication of sample quality that can help determine whether a single-cell genomics workflow should proceed.
Figure 2. Comparison of trypan blue and acridine orange / propidium iodide methods for assessing cell viability.
Although samples stained with AO/PI can be assessed manually using a fluorescence microscope, automated cell counters provide more consistent results. Not only do automated systems remove user bias from the cell counting process, but they also enable the adoption of standardized methods to improve reproducibility and streamline workflows. Fluorescence-based cell counters, especially, promise to enhance single-cell sequencing applications, where they offer greater flexibility than counters providing brightfield measurements alone.
As well as the capacity for fluorescence-based counting, another feature to look for when selecting an automated cell counter for single-cell genomics research is a low volume requirement. Since sample material is often available in only limited supply, instruments that allow a low analysis volume (5–10 µL) to be used for multiple quality control purposes can be advantageous. The capacity for slide-free operation is also important since it promotes greener working practices; however, it should be noted that having the option to include a slide if desired means that material can be transferred to a high magnification microscope for analysis of nuclear integrity.
The advantages of AO/PI staining over Trypan Blue are readily apparent when working with challenging sample types such as tissue lysates. These contain large quantities of debris that can be mis-identified as viable cells by Trypan Blue-based methods, and may also include partial nuclei that Trypan Blue is unable to distinguish from intact structures. In a direct comparison of the two methods for counting isolated nuclei from mouse brain using an automated cell counter, the viability using Trypan Blue was calculated at 54%, whereas AO/PI staining provided a viability measurement of just 1.8% that was confirmed by a manual count. The Trypan Blue method also failed to count a proportion of the nuclei, highlighting the importance of using AO/PI staining to achieve the precision necessary for single-cell genomics applications.
Figure 3. Comparison of viability counts using either brightfield (trypan blue) or fluorescence (acridine orange / propidium iodide) methods.
The CellDrop™ is an automated cell counter that allows analysis of low sample volumes and can be used to accurately and reproducibly count nuclei for single-cell genomics applications. It also reduces the environmental impact of research by eliminating the need for disposable slides. To learn more, visit denovix.com