2-COLOR FLOW CYTOMETRIC ANALYSIS OF INTRAERYTHROCYTIC MALARIA PARASITE DNA AND SURFACE MEMBRANE-ASSOCIATED ANTIGEN IN ERYTHROCYTES INFECTED WITH PLASMODIUM-FALCIPARUM

A method for fixation of Plasmodium falciparum infected erythrocytes and solubilization of the erythrocyte membrane with detergent was developed. This method was applied to two-color flow cytometric analysis of both intraerythrocytic (IE) malaria DNA and parasite-derived antigen on the erythrocyte surface membrane. Infected erythrocytes were fixed with 0.025% glutaraldehyde followed by treatment with 1% saponin to gain access to intramembranous components and allow antibody to interact with antigen. DNA of IE parasite was subsequently stained with propidium iodide. Using this procedure cell morphology was well preserved with excellent parasite DNA staining. Using anti-malaria antibodies which recognize ring-infected erythrocyte surface antigen (Pf155/RESA), we observed that glutaraldehyde-fixed saponin treated infected erythrocytes exhibited a variable immunofluorescence intensity as assessed by both flow cytometry and fluorescence microscopy. Ring-infected cells displayed strong immunofluorescence staining, whereas a weak signal was detected on cells containing schizonts. Simultaneous measurement of parasite DNA and antigen in the infected erythrocyte membrane can facilitate the study of antigen expression in the cell membrane in association with development of IE parasites.