Staining of cellular mitochondria with LDS-751

We have found the dye LDS-751 to bind almost exclusively to mitochondria wh en incubated with viable, nucleated cells. Treatment of cells with the nucl ear stain acridine orange and LDS-751 revealed little colocalization when t he cells were examined by confocal microscopy. Staining with the dye rhodam ine 123, which is known to bind polarized mitochondria, was virtually ident ical to the pattern observed with LDS-751. This staining pattern was observ ed to be consistent over a range of 0.02-20 mug/ml LDS-751 and was consiste nt between both fibroblasts and monocytes. Depolarization of mitochondria w ith the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) dramatically reduced both LDS-751 st aining, and rhodamine 123 fluorescence. Taken together, these results sugge st that LDS-751 is excluded from the nucleus and binds the polarized membra nes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate bet ween leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing. (C) 2001 El sevier Science B.V. All rights reserved.