Izumiyama Shinji, Omura Mako, Takasaki Tomohiko, Ohmae Hiroshi, Asahi Hiroko
Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
Exp Parasitol. 2009 Feb;121(2):144-50. doi: 10.1016/j.exppara.2008.10.008. Epub 2008 Nov 5.
Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.
用于测试对疟原虫促生长和抗疟疾功效的可靠分析技术非常重要。流式细胞术（FCM）提供了利用核酸染色研究疟原虫红细胞内生长发育阶段的可能性。为了分析恶性疟原虫的生长情况，引入了SYBR Green I作为一种嵌入染料，与FCM一起用于氩离子激光的488nm谱线。对采用FCM的程序，包括固定剂、染料浓度、稀释缓冲液和染色时间进行了优化，以简化该方法。本文所述的FCM可根据DNA含量对不同阶段的疟原虫血症和疟原虫进行定量。通过FCM和吉姆萨方法估计的被寄生红细胞比例与通过寄生虫乳酸脱氢酶测定的结果一致。该方案扩展到裂殖子计数，作为寄生虫生长抑制的一种敏感检测方法。
