Journal:

Article Title: A determination of the steady state lysosomal pH of bloodstream stage African trypanosomes

doi: 10.1016/j.molbiopara.2008.02.003

Figure Lengend Snippet: Panel A. Subcellular localization of endocytosed TL:OG488. Bloodstream trypanosomes were loaded with conjugate as described in the text. Cells were fixed, stained with anti-p67 and appropriate secondary reagent, imaged and deconvolved as described in [3]. Merged DAPI/DIC, green channel (TL:OG488), red channel (p67), and three channel merged fluorescence images are presented. Yellow staining in the 3x merged image indicates colocalization of probe with lysosomal p67 marker (arrowhead). Panel B. Flow cytometry scatter plots of DAPI vs OG488 fluorescence are presented for three representative values across the full range of pH(ext) titration, with (bottom) or without (top) 5 mM ammonium chloride. DAPI-negative, OG488 positive cells are in the lower right quadrant, and % viable (DAPI-negative) cells are indicated. 104 events in this quadrant were scored for every mean fluorescent intensity measurement (MFI). Panel C. A representative pH(int) determination is presented. MFI values are plotted as a function of pH(ext) for control (λ) and ammonium chloride treated (ν) samples. Curve fitting (linear regression) and statistical analyses were performed with Prism 4 software (GraphPad Software, Inc., San Diego CA). The calculated pH(int) value for this independent experiment is indicated. Panel D. As in Panel C except cells were treated with bafilomycin A1 (2 μM) for 30 minutes prior to, as well as during, the uptake procedure.

Article Snippet: Mean green fluorescent intensities (λ ex 488 nm/λ em 524 nm) were determined by gating on viable DAPI-negative cells.

Techniques: Staining, Fluorescence, Marker, Flow Cytometry, Titration, Control, Software