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FilenameFormatSizeDescription
bit25127-sm-0001-SuppTab-S1.tif6863KTable SI. Description of fluorogen-activating-proteins (FAPs) used as affinity reagents in this study.
bit25127-sm-0002-SuppFig-S1.tif13002KFigure S1. Description of fluorogens used in this study: names, abbreviations, chemical structures, and excitation/emission wavelengths.
bit25127-sm-0003-SuppFig-S2.tif18161KFigure S2. Flow-cytometry gating strategies using control samples. A: Gating for cell surface HA-epitope on live U937 cells. Leftmost dot plot shows forward and side scatter profiles. Middle plot shows (MG-FAP)-ZZ channel and cell surface TO-FAP channel. Rightmost plot shows histogram of (MG-FAP)-ZZ channel. B: Gating for cell surface native marker on live HEK293 cells. Leftmost dot plot shows forward and side scatter profiles. Middle plot shows propidium iodide channel. Rightmost plot shows histogram of live cells for the (MG-FAP)-ZZ channel. C: Gating for intracellular EEA1 protein in fixed and permeabilized U937 cells. Left dot plot shows forward and side scatter profiles. Right dot plot shows histogram of (MGFAP)-ZZ channel.
bit25127-sm-0004-SuppFig-S3.tif12993KFigure S3. Fluorimetry graphs of in vitro fluorescence activity from different FAP-reagents. A: Measurements were performed using 500 nM (DIR-FAP)-ZZ reagent and 1 μM DIR fluorogen in PBS. B: Measurements were performed using 500  Measurements were performed using 500 nM (MG-FAP)-PrG reagent and 1 μM MG-2p fluorogen in PBS. All samples were assayed in triplicates of 200 μl volume.
bit25127-sm-0005-SuppFig-S4.tif6998KFigure S4. FAP-reagents in vitro IgG-binding. SDS–PAGE gel electrophoresis images of rabbit-IgG chromatography using (DIR-FAP)-ZZ, (TO-FAP)-ZZ, and (MG-FAP)-PrG.
bit25127-sm-0006-SuppFig-S5.tif4919KFigure S5. Micrographs comparing 2-steps versus 1-step surface labeling. For 2-steps labeling, the “Materials and Methods” protocol was performed using live U937 cells. For a 1-step labeling, (MG-FAP)-ZZ reagent to antibody were pre-incubated together (10 to 1 molar ratio) for 5 min on ice, then used for simultaneous labeling of live U937 cells. Cells were visualized using 100 nM of MG-2p fluorogen using a 40× objective.
bit25127-sm-0007-SuppFig-S6.tif9418KFigure S6. Determining FAP-ZZ reagent stability over time. Live U937 cells expressing surface HA-epitope were labeled with anti-HA antibody and (MG-FAP)-ZZ. The cells were rested on ice and imaged at 1, 2, 4, and 6 hours using 100 nM of MG-2p fluorogen with a 40× objective.
bit25127-sm-0008-SuppFig-S7.tif582KFigure S7. Non-specific binding assessment comparing (MG-FAP)-ZZ and (MG-FAP)-PrG affinity reagents. Live U937 cells expressing HA-epitope at the surface were labeled with anti-HA antibody and either (MG-FAP)-ZZ or (MG-FAP)-PrG reagents in increasing concentrations. Flow-cytometry histograms reflect a minimum of 10K live events in the presence of 100 nM MG-2p fluorogen.
bit25127-sm-0009-SuppFig-S8.tif383KFigure S8. Fluorescence intensities comparison between FAP-based versus conventional affinity reagents. A: A flow-cytometry histogram from live U937 cells labeled against a cell surface HA-epitope. A minimum of 10K live cell events were collected in the presence of 100 nM MG-2p fluorogen. B: Mean pixel intensities from micrographs of live HEK293 labeled at the cell surface using equal settings comparing (MGFAP)-ZZ versus Alexa-647 secondary (n = 15 images each group), or (TO-FAP)-ZZ versus FITC secondary (n = 20 images each group). Fluorogen concentrations were 100 nM of MG-2p or TO1-2p.
bit25127-sm-0010-SupData-S1.docx17KSupplementary Materials and Methods.

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