Figure 5

Comparison of three methods for quantifying trypanosomatid DNA. The DNA content of exponentially growing procyclic Trypanosoma brucei and promastigote Leishmania mexicana was analyzed by three different methods. (A) Histograms of total DNA content of cells as measured by flow cytometry of propidium iodide (PI)-stained and RNaseA-treated cells (n = 10,000). Kinetoplast and nuclear DNA cannot be quantified separately by this method. (B) Histograms of nuclear and kinetoplast DNA content cells as measured by quantification of signal from manually outlined kinetoplasts and nuclei in 4', 6-diamidino-2-phenylindole (DAPI) fluorescence images of RNaseA-treated cells (T. brucei n = 552, L. mexicana n = 932). (C) Histograms of nuclear and kinetoplast DNA content as measured by quantification of signal within the cell boundary from the kinetoplast and nuclear images generated from color deconvolution of micrographs of cells stained with DAPI and PI (T. brucei, n = 386) or SYBR green (L. mexicana n = 790). Flow cytometry and nuclear DNA histograms were further analyzed by fitting of a model (black line) of the G1 (red), S (purple) and G2 (green) phases of the cell cycle, the results of which are shown in Table 1.