Accurate quantification of cellular samples is essential for a broad range of applications, from routine cell culture maintenance to complex techniques like single-nucleus RNA sequencing (snRNA-seq). Yet traditional methods for assessing cell concentration and viability often involve a series of manual processes that can damage sensitive cell types and risk introducing user-to-user variability. An all-in-one disposable slide solution that integrates cell sampling and staining into a single step offers an efficient solution for precise, reproducible cell counting.
One of the best-known methods for cell counting involves loading a single-cell suspension into a hemocytometer and counting the cells by eye using a brightfield microscope. To determine cell viability, trypan blue is added to the sample, where it is taken up by dead cells due to the permeability of their damaged membranes.
Although manual cell counting is simple and inexpensive, it is also incredibly slow and tedious, with significant potential for human error. To address these limitations, automated cell counters have been developed that provide high-throughput, imaging-based quantification, including statistical information on cell size—a key indicator of cell quality.
Based on the same principles as hemocytometry, automated cell counters encompass early-generation models that operate in brightfield mode and newer instruments, such as the LUNA-FX7™ Automated Cell Counter from Logos Biosystems, that additionally feature fluorescence capabilities for greater counting accuracy.
Dual staining with acridine orange (AO) and propidium iodide (PI) is one of the most widely used methods for fluorescence-based cell counting. AO is a cell membrane permeable dye that emits a distinctive green fluorescence once bound to DNA, while PI can only enter cells with damaged membranes, where it emits red fluorescence upon DNA binding. In non-viable cells, the PI signal absorbs the AO signal due to Förster resonance energy transfer (FRET), such that live cells only fluoresce green and dead cells only fluoresce red. Importantly, because AO/PI will only stain nucleated cells, cellular debris and non-nucleated cells are excluded from the analysis.
When staining cells with AO/PI for evaluation using a fluorescent cell counter, the first step is to generate a single-cell suspension. Next, a small aliquot of the cell suspension is mixed with the AO/PI solution according to the manufacturer’s instructions. The stained cells are then loaded into the instrument, usually in a disposable slide, and the counting process is initiated.
A drawback of this method is that cell sampling and cell staining are performed as two distinct steps, which increases the risk of damaging sensitive cell types and can accentuate user-to-user variability. Additionally, more waste is generated as a result of performing multiple pipetting actions, which can impact a laboratory’s green credentials.
To overcome the challenges associated with traditional AO/PI staining workflows, Logos Biosystems has developed an all-in-one disposable cell counting slide for the LUNA-FX7 Automated Cell Counter that integrates cell sampling and cell staining. Known as the SpectraSlide® AP-1, it is preloaded with AO and PI, which dissolve instantly upon contact with liquid to ensure immediate and uniform cell staining.
For ease of use, the SpectraSlide AP-1 features an intuitive “click–dip–release” sample loading method, whereby the user simply depresses a rubber dome, inserts the slide into the sample, and releases the pressure to aspirate the cells. The SpectraSlide AP-1 is then placed in a slide bed and loaded into the LUNA-FX7, where it is allowed to settle for a brief (30 second) interval prior to automated cell concentration and viability analysis. In combination, the click mechanism and slide bed serve to prevent cross-contamination, keeping usability and quality control in check.
A major advantage of integrated cell sampling and handling is that it increases counting accuracy by requiring just a single sample handling step. In turn, this helps to improve workflow efficiencies and decrease waste, as well as saves money. The SpectraSlide AP-1 has been evaluated for use with various sample types, including common cell lines such as CHO-K1, HEK293, HL60, and Jurkat, as well as splenocytes and isolated nuclei, whose small size often presents challenges for image-based cell counters.
To learn more about the SpectraSlide AP-1, including validation data and application notes, visit logosbio.com/spectraslide