Journal: eLife

Article Title: Cell surface localisation of GPI-anchored receptors in Trypanosoma brucei

doi: 10.7554/eLife.107191

Figure Lengend Snippet: Initial binding is to the cell surface and internalisation is detected within 60 s. Cultures of single (1-GPI) or double (2-GPI) GPI-anchored TfR expressing BES7-VSG221 and BES1-VSG221 cells were supplemented with 500 nM Alexa568-labelled holo-transferrin (Tf A568 ) and incubated for 15, 60, or 600 s and fixed by addition of formaldehyde for 5 min. Following washing with PBS containing 1% bovine serum albumin cells were visualised under Zeiss Axio Imager.Z2 with ×100 objective. Images in all these experimental time points for both 1- and 2GPI TfR cells were captured with same intensity of excitation and exposure time. ( A ) Images showing the location of bound or endocytosed transferrin in representative cells in populations and at time points as indicated. Scale bar is 5 µm. Images were processed with ZEN Blue 3.4 software without any deconvolution. Larger numbers of cells are shown in . ( B ) The variation between individual cells in transferrin binding and uptake and endocytosis shown by measurements of total fluorescence intensity of Tf A568 in BES7 VSG221 1- and 2-GPI TfR cells at 15, 60, and 600 s. Measurements were obtained using Zen Blue 3.4 software and normalising the background fluorescence . Scatter plots of measurements of individual cells are shown for 1-GPI (grey) and 2-GPI (purple) TfRs. The average and standard deviation are shown for each cell line. ( C ) Tf A568 fluorescence intensity distribution across transverse sections of randomly selected cells was measured using ImageJ software. The plots are shown for both BES7 1- and 2-GPI cell lines, showing the pattern of individual cells in different colours at 60 and 600 s. ( D ), ( E ) and ( F ) same as ( A ) ( B ) and ( C ) but with BES1-VSG221 1- and 2GPI TfR expressing cells .

Article Snippet: Bovine holo-transferrin (Thermo Fisher) was dialyzed against PBS and labelled with Alexa fluor 568 NHS ester (Thermo Fisher) following the manufacturer’s protocol.

Techniques: Binding Assay, Expressing, Incubation, Software, Fluorescence, Standard Deviation

Journal: eLife

Article Title: Cell surface localisation of GPI-anchored receptors in Trypanosoma brucei

doi: 10.7554/eLife.107191

Figure Lengend Snippet: ( A ) Clustal W alignment of the polypeptide sequences of cow and dog transferrin. ( B ) Cultures were supplemented with 500 nM Alexa568-labelled canine holo-transferrin and incubated for 60 s and fixed in culture by addition of formaldehyde to 1%. Following washing with PBS containing 1% bovine serum albumin cells were visualised under Zeiss Axio Imager.Z2 with x100 objective. Images for both BES7 VSG221 1- and 2-GPI TfR cells were captured with same intensity of excitation and exposure time. Images showing the absence of any bound or endocytosed canine transferrin in around 40 cells in population of both 1- and 2GPI TfR cells. Scale bar is 20 µm. Images were processed ZEN Blue 3.4 software.

Article Snippet: Bovine holo-transferrin (Thermo Fisher) was dialyzed against PBS and labelled with Alexa fluor 568 NHS ester (Thermo Fisher) following the manufacturer’s protocol.

Techniques: Incubation, Software