Kinetoplast and nuclear DNA staining in Leishmania mexicana

Kinetoplast and nuclear DNA in kinetoplastids bind different DNA stains with different affinities. Staining of Leishmania mexicana promastigotes was optimised for five DNA stains (DAPI, propidium iodide, SYTO16, DRAQ5 and hoechst 33342). Maximum brightness (maximum pixel intensity) and total signal (sum pixel intensity) of the kinetoplast and nucleus were quantified from widefield epifluorescence micrographs for each stain. Two stains were analysed from methanol fixed cells mounted in glycerol DABCO: DAPI and propidium iodide. The others were analysed from live cells in PBS. Propidium iodide staining included an RNase A digestion. This resource lets the relative contribution of kinetoplast and nuclear DNA be determined from signal intensity data from fluorescence micrographs and flow cytometry. This is a useful resource if working with the methods in one of my previous papers; see links.

动质体目原生生物（kinetoplastids）的动基体（kinetoplast）与核DNA对不同DNA染色剂的结合亲和力存在差异。本研究针对五种DNA染料（DAPI、碘化丙啶、SYTO16、DRAQ5及Hoechst 33342），优化了墨西哥利什曼原虫（Leishmania mexicana）前鞭毛体的染色方案。针对每种染料，我们从宽场落射荧光显微图像中定量了动基体与细胞核的最大亮度（像素最大强度）及总信号（像素强度总和）。其中DAPI与碘化丙啶两种染料采用甲醇固定后以甘油-DABCO封片的细胞进行分析，其余三种染料则直接对磷酸盐缓冲液（PBS）中的活细胞进行成像分析。碘化丙啶染色流程中加入了核糖核酸酶A（RNase A）消化步骤。该数据集可通过荧光显微图像与流式细胞术（flow cytometry）获取的信号强度数据，实现动基体DNA与核DNA相对贡献占比的定量测定。若需沿用本团队此前发表论文中的相关实验方法，可参考文末链接。