Pattanapanyasat K, Webster H K, Udomsangpetch R, Wanachiwanawin W, Yongvanitchit K
Thalassemia Center, Faculty of Graduate Studies, Siriraj Hospital, Bangkok, Thailand.
Cytometry. 1992;13(2):182-7. doi: 10.1002/cyto.990130212.
A novel fixative and permeabilization method is described which allows simultaneous 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 immunolabeled 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 intraerythrocytic 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 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 cell membrane proteins in association with development of intracellular malaria parasites.
本文描述了一种新型的固定和通透方法，该方法能够同时通过流式细胞术检测红细胞膜抗原和细胞内疟原虫。为了说明该方法，将阵发性夜间血红蛋白尿患者的红细胞用恶性疟原虫感染，并维持在同步红细胞培养中。将感染的红细胞用针对补体调节蛋白衰变加速因子（DAF）的抗体进行免疫标记，随后在多聚甲醛中固定，然后在磷酸盐缓冲盐水中用戊二醛固定。最后，用碘化丙啶对红细胞内寄生虫的DNA进行染色。使用该技术，细胞形态得到了很好的保存，未观察到细胞聚集，并且获得了高质量的间接免疫荧光和寄生虫DNA染色，非特异性标记可忽略不计。同时测量寄生虫DNA和红细胞膜决定簇使得研究与细胞内疟原虫发育相关的红细胞膜蛋白改变成为可能。
