Peripheral blood mononuclear cells (PBMCs) are often processed to separate from whole blood by density gradient centrifugation. Those cells are consist of lymphocytes (T cells, B cells, NK cells) and monocytes, commonly used in the field of immunology, cell therapy, infectious disease and vaccine development. Monitoring and analyzing viability and concentration of PBMC is crucial for the clinical laboratories, basic medical science research and immune cell production.
Fig 1. Isolated PBMC from fresh blood with Density gradient centrifugation
AOPI Dual-fluoresces counting is the assay type used for detecting cell concentration and viability. The solution is 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.