Department of Pharmaceutical Sciences, University of Puerto Rico, San Juan, Puerto Rico.
Cytometry A. 2011 Nov;79(11):965-72. doi: 10.1002/cyto.a.21119. Epub 2011 Aug 25.
Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. © 2011 International Society for Advancement of Cytometry.
流式细胞术是一种潜在有效的方法，可用于定量实验性疟疾感染和抗疟药物敏感性测定中的疟原虫血症，方法是使用伯氏疟原虫等啮齿动物疟疾模型。在这项研究中，我们使用两种红色 DNA 结合荧光染料，罗丹明 800（R800）和 LD700，来测量感染伯氏疟原虫的小鼠全血样本中的疟原虫血症水平。血液样本用 RNAse A 处理以消除 RNA 衍生的信号。碘化丙啶，可同时染 DNA 和 RNA，用作阳性对照。R800 和 LD700 确定的疟原虫血症水平与血涂片显微镜分析和使用 Hoechst 33258 的流式细胞术计算的水平相当。RNAse 处理不影响这些测量。我们还使用 R800 或 LD700 来量化感染 GFP 表达的伯氏疟原虫系的小鼠中的疟原虫血症，以将 DNA 染色确定的疟原虫血症水平与使用 GFP 荧光作为替代测量的寄生虫数量相关联。发现使用这些染料的流式细胞术确定的水平与 GFP 表达测量的水平之间存在正相关。当将流式细胞术确定的疟原虫血症与常规显微镜确定的疟原虫血症进行比较时，也得到了相似的结果。使用 R800 或 LD700 染色检测感染的红细胞的检测限分别为 0.1%和 0.15%。这项研究表明，基于红色激光的流式细胞术使用 R800 或 LD700 可有效定量感染的红细胞中的疟原虫血症水平。此外，该方法具有不需要 RNAse 预处理的优点，并且可以分析比常规显微镜测量的更多的细胞来测量寄生虫负担。2011 年国际细胞分析促进协会。
