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

A method for fixation of Plasmodium falciparum infected erythrocytes a nd solubilization of the erythrocyte membrane with detergent was devel oped. 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 ga in access to intramembranous components and allow antibody to interact with antigen. DNA of IE parasite was subsequently stained with propid ium iodide. Using this procedure cell morphology was well preserved wi th excellent parasite DNA staining. Using anti-malaria antibodies whic h recognize ring-infected erythrocyte surface antigen (Pf155/RESA), we observed that glutaraldehyde-fixed saponin treated infected erythrocy tes exhibited a variable immunofluorescence intensity as assessed by b oth flow cytometry and fluorescence microscopy. Ring-infected cells di splayed strong immunofluorescence staining, whereas a weak signal was detected on cells containing schizonts. Simultaneous measurement of pa rasite DNA and antigen in the infected erythrocyte membrane can facili tate the study of antigen expression in the cell membrane in associati on with development of IE parasites.