MDCs were isolated from the bone marrow of WT MPO+/+ C57BL/6 (Taconic Biosciences, Rensselaer, NY) or age-matched syngeneic C57BL/6 MPO-deficient (MPO−/−, MPOtm1Lus; the Jackson laboratory, Bar Harbor, ME, USA) animals. Intracardiac heart puncture was done before animal euthanasia by cervical dislocation. Femurs and tibias were removed from the animals and stored in cold isolation buffer [PBS with 2% FBS and 1× penicillin/streptomycin (Pen/Strep)], and the ends of each bone were removed to expose the bone marrow. Femurs and tibias were placed in a 0.5-ml Eppendorf tube with a hole in the bottom punctured by an 18G needle and then placed in a 1.5-ml Eppendorf tube and centrifuged for 1 min at 1 to 2000 rpm to pellet the bone marrow cells to the bottom of the 1.5-ml Eppendorf tube. The bones were then flushed with cold isolation buffer to further remove any residual bone marrow cells. The bone marrow cells were resuspended with isolation buffer, filtered through a cell strainer, and centrifuged again at 1000 to 2000 rpm for 2 min. The cell pellet was resuspended in 1 ml of isolation buffer. MDCs were isolated using a neutrophil isolation kit (Miltenyi Biotec, Auburn, CA). CD11b+Ly6G+ MDCs were quantified by flow cytometry in which 5 × 105 cells before and after isolation were labeled with 5 μl of CD11b-FITC (fluorescein isothiocyanate) (Miltenyi Biotec, Auburn, CA) and 5 μl of anti–Ly6G-APC (Miltenyi Biotec, Auburn, CA) in 45 μl of flow buffer [PBS (pH 7), 0.5% bovine serum albumin (BSA), and 2 mM EDTA] for flow cytometry analysis at the South Campus Flow Cytometry and Cell Sorting Core at UT MD Anderson Cancer Center. MPO activity was assessed by luminol imaging (7) of 5 × 104 isolated myeloid cells added to 100 μM luminol in colorless DMEM with or without 50 μM PMA. Cells were imaged immediately for 20 min using the IVIS 100 bioluminescence imaging system (PerkinElmer/Caliper Life Sciences, Waltham, MA) at 37°C under 5% CO2 flow. Typical acquisition parameters were as follows: acquisition time, autoexposure; binning, 8; FOV, 15 cm; f/stop, 1; filter, open; image-image interval, 5 min; total number of acquisitions, 4. Cells (2 × 104) of each B16F10 reporter cell line (NF-κB→FLuc, pκB5→FLuc, and pκB5→IκBα-FLuc) were seeded in a 96-well black-walled plate. Twenty-four hours later, d-luciferin (150 μg/ml) in phenol-free DMEM with 10% FBS was added to all wells and allowed to equilibrate for 30 min at 37°C in a humidified 5% CO2 atmosphere prior the addition of 2 × 105 isolated myeloid cells. BLI reporter levels were imaged using the IVIS 100 bioluminescence imaging system at 37°C under 5% CO2 flow. Typical acquisition parameters were as follows: acquisition time, autoexposure; binning, 8; FOV, 15 cm; f/stop, 1; filter, open; image-image interval, 5 min; total number of acquisitions, 3 segments for basal BLI levels, 1 segment after MDC addition, and 24 segments after TNFα addition. For PMA-stimulated MDCs, 50 μM PMA was added to MDCs for 2 min in 500 μl of myeloid cell media (colorless DMEM with 10% FBS and 1× Pen/Strep), and 2 × 105 PMA-stimulated MDCs were added to B16F10 reporter cells. Where indicated, TNFα (0.1 μg/ml) was added to coculture wells 5 min after the addition of MDCs. Bioluminescence photon flux (photons/s) data were analyzed by ROI measurements in Living Image 4.5 (PerkinElmer/Caliper Life Sciences, Waltham, MA). Raw data were imported into Excel (Microsoft Corp., Redmond, WA), averaged in each experiment, normalized to initial (t = 0) values (fold initial), and normalized to vehicle-treated controls (fold control). Quantification of the amplitude and timing of IκBα degradation and resynthesis or NF-κB and κB5 synthesis was carried out in Excel. Fold initial, fold control reporter dynamic plots were generated in GraphPad Prism (GraphPad Software Inc., La Jolla, CA). After each MDC isolation, the percentage of CD11b+Ly6G+ cells was quantified by flow cytometry. Using the percentage of CD11b+Ly6G+ cells (which were considered the myeloid-derived population), the number of MPO+/+ and MPO−/− MDCs added to the coculture studies was calculated and all fold initial, fold control dynamic plots were normalized to 1000 MDCs. For neutrophil coculture microscopy experiments, 1 × 106 of B16F10 NF-κB→FLuc reporter cells were seeded onto a glass-bottom 35-mm dish 24 hours before imaging studies. Isolated MDCs (MPO+/+ or MPO−/−) (1 × 106) were labeled with 5 μl of anti–Ly6G-APC following the flow cytometry labeling protocol described above. Anti–Ly6G-APC–labeled MDCs (1 × 105) were added to each dish for 1 hour at 37°C in a humidified 5% CO2 atmosphere. After 1 hour, all media was removed from the dish and replaced with d-luciferin (150 μg/ml) in phenol-free DMEM with 10% FBS, which was allowed to equilibrate for 30 min at 37°C in a humidified 5% CO2 atmosphere. Before microscopy imaging, 0.25 μl of Hoechst 33342 stain (10 mg/ml) was added to stain nuclei. A Nikon TiE inverted LSM microscope was customized to acquire multimodal bioluminescence and fluorescence images. An air-cooled 4-megapixel (−70°C or less) back-illuminated camera (Andor) was used for BLI acquisition. BLI acquisition parameters were as follows: sensor gain set to 4×, read speed of 50 kHz, and binning between 1 and 2 depending on the acquisition using a 20-min exposure. Fluorescence was imaged using the Nikon TiE inverted LSM C2 confocal system and the following image acquisition parameters: 4.8 speed on a 20× water objective using laser lines of 405 nm (Hoechst excitation), 488 nm (dendra2, GFP excitation), and 640 nm (APC excitation). The FOV was slightly different between BLI images and confocal images because of the use of two different cameras for each acquisition. Therefore, when BLI images with confocal images were overlaid and compared, only FOVs found in both images were analyzed. Quantification of NF-κB transcriptional activation based on NF-κB→FLuc bioluminescence was as follows. Using the NIS-Elements AR Analysis software, cell count analysis occurred on the 640-nm confocal image to identify all APC-labeled MDCs using the following parameters: threshold minimum of 300, size minimum of 9 μm, and circularity minimum of 0.2. Using the overlaid confocal image at 405, 488, and 640 nm, ROIs were drawn around B16F10 cells that were in close proximity, either in contact or within a distance of 5 μm, of all identified MDCs. These ROIs were copied and pasted onto the corresponding bioluminescence microscopic images, and photon flux counts of B16F10 cells in close proximity to MDCs ROIs were measured. Similarly, a minimum of 20 ROIs (or twice as many ROIs identified for B16F10 cells in contact with MDCs, whichever was greater) were drawn randomly around B16F10 cells that were distant (greater than 5 μm) from MDCs on the confocal images. These ROIs were copied and pasted onto the corresponding bioluminescence microscopic images, and photon flux counts of B16F10 cells distant from MDCs were measured. All ROI bioluminescence counts quantified were normalized to ROI area.

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