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Table 1
Table 2

strains harboring pCC vectors containing the roGFP2-based probes ( T By Alexander Wang Woman Laceup Stretchmodal Jersey Midi Dress Red Size M Alexander Wang qNvNQuXTtW
) were cultured in LB liquid medium with 200 µg/mL of ampicillin at 37°C overnight. The optical density at 600 nm (OD) was measured and the bacterial suspension was diluted to an OD of 0.1 with fresh medium and cultured at 37°C for~2 hr until an OD of 0.5–0.8 was reached. The expression of roGFP2-based probes was then induced with 100 µM IPTG and the culture was incubated at 20°C overnight. These bacterial cells, now containing roGFP2-based probes, were then washed twice in 40 mM HEPES buffer (pH 7.4) and re-suspended in 1 mL HEPES buffer to a final OD of 0.3. The fluorescence intensity was measured in an FP-8500 spectrofluorometer (Jasco, Tokyo, Japan). The emission wavelength was fixed at 510 nm and excitation wavelength was scanned from 350 to 500 nm. Bandwidths of excitation and emission were set to 5 nm. The cell suspension in the cuvette was continuously stirred with a magnetic stir bar and the temperature of the temperature controller EHC-813 (Jasco) was set to 25°C. Fluorescence excitation ratios (405/488 nm) were used as measurement of probe oxidation ( Arias-Barreiro et al., 2010 ; Preowned Bobby trainers Isabel Marant IXLicO
). Oxidation with 100 µM aldrithiol-2 (AT-2, Sigma-Aldrich,St.Louis,MO) and reduction with 50 mM dithiothreitol (DTT, Sigma-Aldrich) were used to fully oxidize and fully reduce the probes, respectively ( BELITE S L FOOTWEAR Lowtops amp; sneakers Diadora svMa1qaPG
).

cells expressing roGFP2-based probes as described above were washed twice in PBS pH 7.4 and resuspended in PBS pH 7.4 with 0.5% FBS to a final OD of 0.1–0.3. FBS was omitted in experiments where no PLB-985 cells were present ( Figure 1J and Womens Lily Ballet Flats Vagabond Z094plb
). Fifty microliters of this suspension were placed in the wells of a black, clear-bottom 96-well plate (Nunc, Rochester, NY). Fluorescence intensity was recorded during every minute for 10 min using the Synergy H1 multi-detection microplate reader (Biotek, Bad Friedrichshall, Germany) at the excitation wavelengths 405 and 488 nm and emission wavelength at 530 nm. Then 50 µL of the selected chemical solution or the respective PLB-985 cell suspension was added to the wells and the fluorescence intensity was monitored for another 2 hr under the same conditions. The signals of fully oxidized and fully reduced probes were obtained by adding 100 µM AT-2 or 50 mM DTT to the bacteria culture at the start of the 2 hr measurement. The fluorescence excitation ratios (405/488 nm) were used as measurement of probe oxidation and all values were normalized to the values obtained for fully oxidized (AT-2-treated) and for fully reduced (DTT-treated) bacteria cultures.

cells expressing roGFP-based probes (as described above) were opsonized by incubation for 30 min at 37°C with 1 mg. mL human immunoglobulin G (Sigma-Aldrich) in PBS pH 7.4, then washed twice in PBS and re-suspended in PBS with 0.5% FBS to an OD of 0.1. 50 µL of opsonized was then added to 50 µL of differentiated PLB-985 cell suspension (10 mL) in PBS with 0.5% FBS. Thus a ratio of 10 bacteria per PLB-985 cell was used to initiate phagocytosis. Cells and bacteria were then co-incubated at 37°C, and phagocytosis was stopped by adding 100 µL of ice-cold PBS at certain time points. Cells were fixed with 4% paraformaldehyde and samples were kept on ice until subjected to flow cytometry. Samples were analyzed using a BD FACSCanto II flow cytometer (Becton, Dickinson and Company) with an argon laser operating at 488 nm using the 530/30 emission filter to detect the fluorescence of the roGFP2-based probes expressed in phagocytized bacteria. For each sample, a total of 10,000 viable cells were counted. The mean fluorescence intensity (MFI) multiplied by the percentage of viable cells that had ingested fluorescent particles was used to evaluate the phagocytic capacity of PLB-985 cells. In some experiments, phagocytosis was inhibited by treatment of differentiated PLB-985 cells with 250 µM cytochalasin D (Sigma-Aldrich) for 30 min at 37°C before co-incubation with opsonized bacteria.

Paraformaldehyde-fixed cells from the phagocytosis assay were centrifuged at 200 for 5 min and resuspended in PBS containing 0.1% Tween 20 and 2.5 µg. mL TRITC-conjugated phalloidin (Sigma-Aldrich). Cells were incubated with phalloidin for 40 min at room temperature, protected from light. Excess phalloidin was removed by centrifugation and cells were resuspended by pipetting up and down in buffered glycerol containing 0.5 µg/mL DAPI (Sigma-Aldrich). Cells were then visualized on a slide in a BX51 fluorescence microscope with a U‐UCD8 condenser, a U‐LH100HGAPO burner, a U‐RFL‐T power supply, a 63X/1.4 NA oil objective and a 450–490 nm excitation/500–550 emission bandpass filter (Olympus, Tokyo, Japan). Images were collected with a CC12 digital color camera and the Cell Imaging Software (Olympus) and composite figures were prepared using ImageJ ( Dress for Women Evening Cocktail Party On Sale Black Cotton 2017 6 JC de Castelbaljac 2017 y6caC
) and Photoshop CS5 (Adobe Systems, San Jose, CA) software.

Differentiated PLB-958 cells were stained with 0.25 µM Celltracker Deep Red (Thermo Fisher Scientific, USA) in RPMI 1680 at 37°C and 5% CO for 30 min, washed once with PBS and diluted in PBS with 0.5% FBS to a final concentration of 10 cells. mL. 1 mL of the cell suspension was poured onto an imaging dish (µ-Dish 35 mm, high, Ibidi, DE). Opsonized roGFP2-Orp1 cells were added with a ratio of five cells to one PLB-958 cell. Fluorescence images were acquired with an LSM 880 ELYRA PS.1 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were acquired in three different channels: channel I: Ex/Em, channel II: Ex/Em, channel III: Ex/Em, bandwidth settings channels I and II: 13 nm, channel III: 59 nm. Individual single channel images were exported using ZEN 2.1 (Zeiss, DE). Ratiometric images were generated with ImageJ 1.51e (National Institutes of Health, USA) as described ( Ruched Pleated Maxi Prom Dress Waterlily Little Mistress Petite 9dsvL
). The image background was corrected using a rolling ball algorithm and images were transferred to 32-bit format. Images were thresholded and converted to binary mask. Images from channel I and channel II were aligned using the ImageJ plugin „MultiStackReg“and ratiometric images were calculated using „Ratio Plus“. Ratiometric images were colored using the „Lookup table“feature from the plugin „NucMed“. The display range of all ratiometric images were adjusted to the same range before converting them into RGB format. For the ratiometric time series assembly, images were smoothed in order to reduce noise. After background subtraction, normalized 405/488 nm ratio image series were calculated and assembled to a movie using Software kindly provided by Fricker (2016 ).

Intracellular oxidation of 2',7'-dichlorodihydrofluorescein (DCFH) to 2',7'-dichlorofluorescein (DCF) by PLB-985 cells was analyzed using the cell permeable derivative 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). PLB-985 cells (10 mL) were pre-incubated with 1.25 µM DCFH-DA (Thermo Fisher Scientific, Waltham, MA) in PBS with 0.5% FBS during 15 min at 37°C. Then 50 µL were placed in the wells of a black, clear-bottom 96-well plate (Nunc). Fluorescence intensity was recorded every 1 min for 10 min using the Synergy H1 multi-detection microplate reader (Biotek) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Cells were then activated by addition of 50 ng · mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) or incubation with 10-fold excess of bacteria. Fluorescence intensity was then recorded every 1 min for 1 hr.

Myeloperoxidase activity in neutrophil-like PLB-985 cells was inhibited by pre-incubation of cells with 500 µM 4-aminobenzoic acid hydrazide (ABAH; Sigma-Aldrich) for 30 min at 37°C prior to subsequent experiments.

All 96-well-plate-based fluorescence measurement experiments were repeated at least three times with biologically independent replicates and the results are expressed as the mean±standard deviation as represented by error bars. Representative data is shown for fluorescence spectroscopy, flow cytometry and microscopy image data.

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Christine Winterbourn
Reviewing Editor

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Neutrophil-generated HOCl is the major factor in the breakdown of the thiol redox potential in phagocytized bacteria" for consideration by eLife . Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gisela Storz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Clare Hawkins (Reviewer #2).

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