AO/PI Fluorescence Assays vs. Trypan Blue

Life scientists leverage a range of fluorescent dyes and assays when it comes to cell viability testing including trypan blue, acridine orange (AO), and propidium iodide (PI). Trypan blue is one of the most common reagents used in culture labs and biosciences to selectively stain dead tissues as part of cell viability testing. AO/PI fluorescence assays, meanwhile, are preferable for samples containing debris and non-nucleated cells.

In this article, DeNovix compares the performance of AO/PI fluorescence assays to trypan blue to assess when fluorescent dyes are better for viability tests.


Assessing Trypan Blue Performance

Trypan blue is an azo dye that works particularly well for distinguishing dead from live cells – but only in samples with minimal debris. It stains dead tissues by penetrating damaged cell membranes and dying them a characteristic blue color but is excluded from live cells which subsequently remain transparent.

Using a CellDropTM Cell Counter from DeNovix, calculating trypan blue viability is simple and intuitive. The dedicated Trypan Blue Application optically counts live/dead cells by identifying and calculating the respective concentration to automatically report cell viability. However, the way trypan blue permeates damaged membranes can prove problematic for some sample types. As a result, it is most suitable for trypsinized cell cultures and other sample types with little cellular debris.

Assessing AO/PI Fluorescence Assay Performance

Samples that have not been subjected to time-consuming purification processes are better suited to tailormade fluorescence assays like AO/PI; a unique combination of acridine orange (a cell-membrane permeable nucleic acid-binding fluorophore) and propidium iodide (a nucleic acid-binding due that cannot penetrate live cells).

The AO/PI Applications on the CellDropTM are optimized for precise cell counting of nucleated cells. By utilizing three distinct fluorescence channels (brightfield, green, and red), the cell counter can acquire accurate imagery of all dead and live cells, even in the presence of debris. Consequently, AO/PI fluorescence assays are the ideal reagent when analyzing primary cells, whole blood samples, irregular/small cells, and so on.

Read Our Full Technical Note on AO/PI Fluorescence Assays Vs. Trypan Blue

If you are looking for a precise fluorescence assay for your cell counting and viability assessments, simply contact a member of the DeNovix team today.