Flow Cytometric Two-Color Staining Technique for Simultaneous Determination ofHuman Erythrocyte Membrane Antigen and Intracellular Malarial DNA

摘要

A novel fixative and permeabilization method is described which allowssimultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immuno labeled with antibodies directed to the complement regulatory protein, decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocyctic parasites was stained with propidium iodide. using this technique, cellular morphology was well preserved, no cell aggregation was observed and high-quality indirect immunofluorescence and cell aggregation was observed and high-quality indirect immunofluorescence and parasite. DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red blood cell membrane proteins in association with development of intracellular malaria parasites.