Cd11b bv711

Mouse blood was collected 1 week after sponge implantation from vena saphena in Ethylenediaminetetraacetic acid (EDTA) tubes. Red blood cells were lysed after which cells were stained with a myeloid panel of antibodies comprising Ly6G-FITC (561105, BD Biosciences), Ly6C-BV605 (563011, BD Biosciences), and CD11b-BV711 (563168, BD Biosciences) for 30 minutes at 4°C. Cells were washed and resuspended in DAPI (Invitrogen) before acquisition on a five laser BD LSR Fortessa and analyzed with DIVA (BD Biosciences).
To determine intracellular ROS levels, cells were stained with Ly6C-BV605 (563011, BD Biosciences) and CD11b-BV711 (563168, BD Biosciences) for 30 minutes at 4°C. After washing, cells were stained with H2-DCFDA and analyzed by flow cytometry as described above.

The hippocampus and parenchyma were separated from the rat brains. After 3 washes, the samples were homogenized on ice into a single-cell suspension with a glass homogenizer and then centrifuged at 1200 rpm for 5 min at 4°C. The cells were resuspended and then incubated with Myelin Removal Beads II (Miltenyi Biotec, Bergisch Gladbach, Germany) to remove the myelin. After that, Fc block was used to block the cells for 30 min on ice before surface staining. Next, the cell surface was stained for 30 min on ice with 50 μl of double distilled water containing 2.5 μl of annexin V PE-cyanine 7, 5 μl of binding buffer, 0.5 μl of Zombie APC-A750 (Biolegend, CA, United States), 1 μl of CD45-BV421 and 1 μl of CD11b-BV711 (BD Biosciences, Franklin Lakes, NJ, United States). The stained cells were washed 3 times, fixed and permeabilized using fixation/permeabilization buffer (BD Biosciences, New Jersey, United States), and then probed with intracellular primary antibodies against GFAP (1:50, BD Biosciences, Franklin Lakes, NJ, United States) and NeuN (1:100, Abcam, Cam-bridge, United Kingdom) in BD Perm/Wash TM buffer. Finally, the cells were transferred into flow tubes and analyzed on a flow cytometer (Beckman Coulter, Atlanta, GA, United States), and 10000 cells per tube were captured for further analysis by CytExpert 2.0 software.

Single cell suspensions extracted from the lungs were stained with a live/dead-Infrared marker (Thermo Fisher) and incubated with monoclonal antibody CD16/CD32 Becton Dickinson Biosciences (BD) to block Fc receptors, before adding a cocktail of directly conjugated mAbs directed against the following surface markers; CD11b-BV711 (M1/70, 563168), Ly6G-PerCp-Cy5.5 (1A8, 560602), CD45-BB515 (30-F11, 564590), SiglecF-PE-CF594 (E50-2440, 562757), CD103-BV421 (M290, 562771), CD11c-BV510 (HL3, 562949), CD3e-BV786 (145-2C11, 564379), CD19-BV605 (1D3, 563148), CD335-BV605 (29A1.4, 560469), and Ly6C-APC (AL-21, 560595) all purchased from BD and FceR1-PE-Cy7 (MAR-1, 25-5898-82), MHC-II-AF700 (M5/114.15.2, 56-5321-82) from Thermo Fisher (ebioscience). Data acquisition was performed with the BD LSR Fortessa (BD) and Diva software, and the analysis was performed with FlowJO X software (TreeStar, Ashland, OR). In initial control experiments, the right lobes were evaluated for the frequency of contaminating peripheral blood mononuclear cells after cardiac PBS infusion by staining with an anti-CD115 mAb (T38-320, 565249, BD), which is uniformly expressed on all circulating blood monocytes (13 (link)) but not on lung monocytes (14 (link)). The frequency of CD115⁺ cells was below 0.01% among the lung cells, arguing that we had little if any blood leukocyte contamination (Figure S1B).