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Allow AO to equilibrate with whole blood for 8 minutes. Analyze by cytometry using a 488 nm laser triggering on green fluorescence for acquisition. Plot on forward vs side scatter, and green vs red fluorescence.
Leukocytes all have roughly equivalent green fluorescence, and are easily distinguished from non-fluorescent erythrocytes. The amount of red fluorescence in the leukocytes depends primarily on the amount of lysosomal granules. AO passively diffuses into intact cells and then into lysosomes. Once in the low pH environment of an intact lysosome, AO becomes positively charged and is unable to re-cross the lysosomal membrane. AO is thereby concentrated in the lysosomal granules, where it precipitates and fluoresces red. Lymphocytes have fewest lysosomes, and little red fluorescence, granulocytes have a large number of lysosomes and fluoresce strongly (but with some variability) red. Monocytes are an easily distinguishable population with intermediate red fluorescence between lymphocytes and granulocytes.
NOTE: to get the full differential effect, the whole blood must be freshly collected. Cells in which the lysosomal pH gradient has broken down will not concentrate AO anymore, and lose their red fluorescence (but still have essentially full green fluorescence from the AO/DNA interaction).
J Natl Cancer Inst 83: 701–707, 1991