Accurate assessment of cell concentration and viability is crucial in the development of cell and gene therapies. However, cell counting accuracy can be compromised by many different variables, potentially wasting patient-derived samples and other precious resources. One way of mitigating this risk is to switch from using traditional cell counting methods to combining all-in-one staining and loading with the advanced imaging capabilities of an automated cell counter. By choosing this approach, users can benefit from superior cell counting accuracy and reproducibility that can help to accelerate cell and gene therapy research.
One of the best-known methods for determining cell concentration and viability involves staining a single-cell suspension with Trypan Blue, manually loading the sample into a hemocytometer, and counting live (unstained) and dead (blue) cells by eye using a brightfield microscope.
Another option is to perform dual staining with acridine orange (AO), a membrane-permeable fluorescent dye that stains live cells green, and propidium iodide (PI), a dye that can only enter cells with damaged membranes, causing them to fluoresce red.
An advantage of AO/PI over Trypan Blue staining is that it excludes both cellular debris and non-nucleated cells from the analysis to provide more accurate results. However, both methods utilize a two-step process for staining and loading that adds time to laboratory workflows and increases the risk of error.
Even when analyzing the same sample, different users can obtain inconsistent results when using traditional cell counting methods. For example, if one user pipets the sample more vigorously than the other, they may generate more cellular debris, while their co-worker may fail to properly disperse cell clumps.
Similarly, the technique used for preparing or mixing viability dyes may be highly variable. While one user might allow reagents to reach room temperature before adding a precisely measured amount to the cells, another might use reagents straight from the fridge without fine-tuning pipetting volumes.
The way in which the glass coverslip is applied to the hemocytometer represents a further source of inconsistency. Failure to generate a tight seal can cause cells to be unevenly distributed or to cluster in specific regions, or may result in the sample drying out due to insufficient volume being loaded.
Additionally, determining what constitutes a cell can be controversial, especially when working with cancer cell lines like AsPC-1 (pancreatic adenocarcinoma) and H-1975 (non-small cell lung carcinoma), which naturally tend to form tight clusters during culture, or with cells that have large and complex morphologies, such as A172 (glioblastoma).
In combination, these small differences can cascade, affecting downstream applications. For this reason, many researchers are choosing to move away from traditional cell counting methods in favor of more automated solutions.
Designed exclusively for use with the LUNA-FX7™ Automated Cell Counter from Logos Biosystems, the SpectraSlide®AP-1 Disposable Cell Counting Device allows for staining and loading cells simultaneously. Users simply press the built-in rubber dome to expel the internal air, dip the SpectraSlide AP-1 into the sample, and then gently release the mechanism to automatically draw a consistent sample volume (approximately ≤ 20 µL) into the internal channel. Here, the highly stable AO/PI coating is instantly dissolved such that live/dead discrimination occurs automatically.
Besides ensuring reproducible sample loading, independent of user technique or pipetting proficiency, the SpectraSlide AP-1 offers several other advantages. These include elimination of the need to prepare or mix viability reagents, thereby avoiding common issues associated with liquid reagent storage such as degradation, contamination, and refrigeration requirements. In addition, the SpectraSlide AP-1 has an integrated safety feature known as SlideBed™, which prevents any residual liquid from seeping into the interior of the instrument.
The performance of the SpectraSlide AP-1 for viability and concentration analysis using the LUNA-FX7 has been validated across a range of experimental settings. These include routine assessment of CHO-K1 and HEK293 cell lines, as well as analysis of more challenging cell types, such as AsPC-1, H-1975, and A172. In all cases, the technology provided fast, consistent results, demonstrating its utility to support a diverse array of workflows.
Traditional cell counting methods have several limitations that can compromise data integrity, potentially resulting in the loss of patient-derived samples or costly cell lines used in cell and gene therapy research. Simultaneously staining and loading samples with SpectraSlide AP-1 technology, prior to analysis with the LUNA-FX7, ensures superior reproducibility in cell counting and can enhance workflow efficiencies for faster time to results. To learn more about SpectraSlide AP-1 technology, visit https://logosbio.com/spectraslide-ap-1-one-touch-innovation-in-cell-counting/