AOPI Dual-fluoresces counting is the assay type used for detecting cell concentration and viability. The solution is a combination of acridine orange (the green-fluorescent nucleic acid stain) and propidium iodide (the red-fluorescent nucleic acid stain). Propidium iodide (PI) is a membrane exclusion dye that only enters cells with compromised membranes, while acridine orange is able to penetrate all cells in a population. When both dyes are present in the nucleus, propidium iodide causes a reduction in acridine orange fluorescence by fluorescence resonance energy transfer (FRET). As a result, nucleated cells with intact membranes stain fluorescent green and are counted as live, whereas nucleated cells with compromised membranes only stain fluorescent red and are counted as dead when using the Countstar® FL system. Non-nucleated material such as red blood cells, platelets and debris do not fluoresce and are ignored by the Countstar® FL software.
Process of Stem Cell Therapy
Figure 4 Monitoring of viability and cell count of mesenchymal stem cells (MSCs) for the use in cell therapies.
Determine MSC viability by AO/PI and Trypan Blue assay
Figure 2. A. Image of MSC stained by AO/PI and Trypan Blue; 2. Comparison of AO/PI and Trypan blue result before and after transport.
The cell refractive index changes, Trypan Blue staining was not that obvious, it is difficult to determine the viability after transport. While dual-color fluorescence allows for staining of live and dead nucleated cells, generating accurate viability results even in the presence of debris, platelets, and red blood cells.