Supplementary MaterialsFigure 1source data 1: Numerical fluorescence spectrometry data represented in Figure 1D. Figure 2A, trace CD11b, Undifferentiated, negative control. elife-32288-fig2-data3.csv (1.6M) DOI:?10.7554/eLife.32288.014 Figure 2source data 4: Numerical flow cytometry data represented in Figure 2A, trace CD11b, DMSO. elife-32288-fig2-data4.csv (1.7M) DOI:?10.7554/eLife.32288.015 Figure 2source data 5: Numerical flow cytometry data represented in Figure 2A, trace CD11b, DMSO, isotype control. elife-32288-fig2-data5.csv (1.5M) DOI:?10.7554/eLife.32288.016 Figure 2source data 6: Numerical flow cytometry data represented in Figure 2A, trace CD11b, DMSO, negative control. elife-32288-fig2-data6.csv (1.6M) DOI:?10.7554/eLife.32288.017 Figure 2source data 7: Numerical flow cytometry data represented in Figure 2A, trace CD11b, DMSO+?IFN. elife-32288-fig2-data7.csv (2.0M) DOI:?10.7554/eLife.32288.018 Figure 2source data 8: Numerical flow cytometry data represented in Figure 2A, trace CD11b, DMSO+?IFN, isotype control. elife-32288-fig2-data8.csv (1.7M) DOI:?10.7554/eLife.32288.019 Figure 2source data 9: Numerical flow cytometry data represented in Figure 2A, trace CD11b, DMSO+?IFN, negative control. elife-32288-fig2-data9.csv (1.7M) DOI:?10.7554/eLife.32288.020 Figure 2source data 10: Numerical flow cytometry data represented in Figure 2B, trace CD16, Undifferentiated. elife-32288-fig2-data10.csv (1.7M) DOI:?10.7554/eLife.32288.021 Figure 2source data 11: Numerical flow cytometry data represented in Figure 2B, trace CD16, DMSO. elife-32288-fig2-data11.csv (1.5M) DOI:?10.7554/eLife.32288.022 Figure 2source data 12: Numerical flow cytometry data represented in Figure 2B, trace CD16, DMSO+?IFN. elife-32288-fig2-data12.csv (1.7M) DOI:?10.7554/eLife.32288.023 Figure 2source data 13: Numerical flow cytometry data represented in Figure 2C, trace CD64, Undifferentiated. elife-32288-fig2-data13.csv (1.6M) DOI:?10.7554/eLife.32288.024 Figure 2source data 14: Numerical flow cytometry data represented in Figure 2C, trace CD64, DMSO. elife-32288-fig2-data14.csv (1.5M) DOI:?10.7554/eLife.32288.025 Figure 2source GW 501516 data 15: Numerical flow cytometry data represented in Figure 2C, trace CD64, DMSO+?IFN. elife-32288-fig2-data15.csv (3.0M) DOI:?10.7554/eLife.32288.026 Figure 2source data 16: Numerical flow cytometry data represented in Figure 2D, trace CD66b, Undifferentiated. elife-32288-fig2-data16.csv (1.5M) DOI:?10.7554/eLife.32288.027 Figure 2source data 17: Numerical flow cytometry data represented in Figure GW 501516 2D, trace CD66b, DMSO. elife-32288-fig2-data17.csv (1.7M) DOI:?10.7554/eLife.32288.028 Figure 2source data 18: Numerical flow cytometry data represented in Figure 2D, trace CD66b, DMSO+?IFN. elife-32288-fig2-data18.csv (1.5M) DOI:?10.7554/eLife.32288.029 Figure 3source data 1: Numerical flow cytometry data represented in Figure 3G, trace Opsonized + 1.25% DMSO. elife-32288-fig5-data2.csv (1.0M) DOI:?10.7554/eLife.32288.046 Shape 5source data 3: Numerical stream cytometry data displayed in Shape 5A, track inside macrophages (van der Heijden et al., 2015). roGFP2 offers several advantages in comparison with available fluorescent redox-sensitive dyes commercially. Like a GFP variant, it could be genetically released into just about any natural system and may become even geared to particular mobile compartments (Dooley et al., 2004; Hanson et al., 2004). Its redox condition, which depends upon the redox condition from the natural system, may then become measured by using an engineered couple of cysteine residues near to the fluorophore. The reversible disulfide relationship formation between these cysteines causes hook conformational modification, which leads to a reversible modification from the protonation position from the fluorophore. The decreased and oxidized type of roGFP2 possess GW 501516 specific fluorescence excitation maxima at 395 and 490 nm consequently, respectively (Dooley et al., 2004). Either the 405/488 nm percentage with laser-based excitation or 390/480 nm percentage on filter-based documenting devices can therefore be utilized to straight determine the probes redox condition (Dick and Meyer, 2010). This ratiometric strategy compensates for variants due to GW 501516 variations in total roGFP2 concentrations, enabling quantitative monitoring. These probes therefore enable compartment-specific real-time ratiometric quantification from the intracellular redox position in prokaryotic aswell as eukaryotic cells (Arias-Barreiro et al., 2010; Bhaskar et al., 2014; Meyer and Dick, 2010; vehicle der Heijden et al., 2015). Right here, we report the GW 501516 usage of three different roGFP2-centered fluorescent redox probes to quantitatively monitor the redox condition of bacteria through the phagocytic procedure. Using the H2O2-delicate roGFP2-Orp1 probe indicated in the cytoplasm of MG1655. This probe was created to measure H2O2 in biological systems specifically. We’re able to express roGFP2-Orp1 stably in from a plasmid (Shape 1A). Using fluorescence spectroscopy, we’re able to determine the oxidation condition from the probe in the cytoplasm using the ratio between the excitation wavelengths of 405 and 488 nm (Dooley et al., 2004; Gutscher et al., 2008; Hanson et al., 2004). Addition of the strong oxidant Aldrithiol-2 (AT-2, 2,2-Dipyridyl disulfide) to the bacterial cells led to full oxidation of the probe, while addition of DTT resulted in full reduction (Figure 1D and G). The exposure to reactive Rabbit Polyclonal to PAK7 species in the phagolysosome could also interfere with the glutathione redox potential (EGSH) within the cell..