Staining of Plasmodium yoelii-infected mouse erythrocytes with the fluorescent dye rhodamine 123

Abstract

The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.