Monitoring the uptake of glycosphingolipids in Plasmodium falciparum-infected erythrocytes using both fluorescence microscopy and capillary electrophoresis with laser-induced fluorescence detection.
The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1-TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique.
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, USA.
David C
DC
Essaka
John
J
White
Pradip
P
Rathod
Colin D
CD
Whitmore
Ole
O
Hindsgaul
Monica M
MM
Palcic
Norman J
NJ
Dovichi
D019075:Electrophoresis, Capillary / Q000379:methods*; D004912:Erythrocytes / Q000469:parasitology*; D005456:Fluorescent Dyes / Q000737:chemistry; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000379:methods*; D010963:Plasmodium falciparum / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry
D005456:Fluorescent Dyes; D006028:Glycosphingolipids; D012235:Rhodamines; C005358:tetramethylrhodamine