Anti cd11b pe

Immediately after sacrifice, the liver tissues were excised and washed with PBS to remove blood. Tissues were minced and digested with 1 mg/mL collagenase type IV (Sigma‒Aldrich) in PBS containing 150 U/mL DNase I for 30 min at 37 °C. The digested liver was suspended in 50 mL of DMEM, and the dissociated cells were filtered through a 100-μm nylon strainer and collected by centrifugation at 300 × g for 4 min. Stromal vascular fraction pellets were resuspended in 0.5 ml of RBC lysis buffer (BioLegend) for 5 min on ice, centrifuged for 5 min at 300 × g, and resuspended in PBS. Cells were incubated with the following fluorescently labeled antibodies for 30 min: anti-F4/80-FITC (eBioscience, 11-4801-82, 1:10) or anti-CD11b-PE (eBioscience, 12-0112-82).
Isolation of LPCs was performed as previously described^{21 (link),26 (link)}. Red cells were lysed with buffer (160 mM NH₄Cl, KHCO₃ containing 0.01% EDTA) and resuspended in Williams E medium (Gibco) with 10% FCS. Following centrifugation at 300 × g for 5 min at 4 °C, the supernatant was collected, washed twice, and resuspended in PBS with 2% FCS for FACS staining. LPCs were stained with anti-LGR5 (Abnova, PAB2591, 1:10) at 4 °C for 30 min and then incubated with Alexa 555-conjugated IgG secondary antibody (Invitrogen, A-21428) at 4 °C for 10 min. The stained cells were analyzed by flow cytometry (BD Bioscience).

Splenocytes isolated from control and TCL1 leukemia-bearing mice were suspended at a density of 4 × 10⁶ cells/ml, incubated with 1.0 µM CM-H₂-DCFDA fluorescent probe (Molecular Probes, Eugene, OR, USA) in PBS at 37 °C for 30 min and then washed with PBS. Next, for distinguishing the population of living granulocytes, the cells were incubated with a Zombie Aqua™ Fixable Viability Kit (BioLegend) for 20 min, washed with PBS and subsequently stained with the following fluorochrome-conjugated antibodies: anti-CD11b-PE (eBiosciences, San Diego, CA, USA), anti-Ly6C-PerCP-Cy5.5 and anti-Ly6G-APC-Cy7 (both from BD Bioscience). After a final wash with PBS, to determine the intracellular ROS levels, the geometric mean fluorescence intensity (MFI) of oxidized CM-H₂-DCFDA in CD11b⁺ Ly6G⁺ cells was analyzed by flow cytometry.

Flow cytometry experimentation was described in a previous publication^{29 (link)}. Anti-CD11b PE (#12–0112) was purchased from eBioscience (San Diego, CA) and the apoptosis was detected by Annexin V antibody and propidium iodide staining according to the manufacturer’s instructions (Annexin V-FITC kit, #556547, BD Bioscience, CA).

The in vitro infected samples were collected into tubes and centrifuged at 1000× g for 10 min. The precipitate was incubated with 2 μl of rabbit polyclonal anti-Histone H3 antibody (citrulline R2 + R8 + R17) (Abcam, Cambridge, UK) for 30 min at room temperature. Following this, 1 μl of anti-CD11b-PE (eBioscience, San Diego, CA, USA) and anti-Ly6G-BV421 (eBioscience) antibodies, and 0.8 μl of AlexaFluor-700-conjugated goat anti-rabbit antibody (Thermo Scientific, Rockford, IL, USA) were added and incubated for 30 min at room temperature in the dark. Then, for whole blood samples, 1 ml of FACS reagent (BD Biosciences, San Diego, CA, USA) was added until red cell lysis was achieved (10−15 min). This was followed by the addition of 1 ml of 2% bovine serum albumin in phosphate-buffered saline (PBS) and centrifugation at 1000× g for 10 min. The supernatant was discarded, and the pellet was resuspended in 300 μl of PBS. Labeled cells were kept on ice and examined with flow cytometry analysis (FACS LX, Beckman-Coulter, Pasadema, CA, USA), which was performed with the CytExpert software (Beckman). The percentage of isotype controls was < 3%. At least 10,000 neutrophil or neutrophil-like populations were collected for each sample. Three independent experiments were performed.

Cells were incubated on ice for 45 min in Fc block in the presence of relevant primary antibodies. The anti-CD11c FITC, anti-CD11b PE, anti-CD103 PECy5, anti-MHCII APC-efluor780, anti-F4/80 efluor450, anti-CD45 BV510, anti-CX3CR1 PECy7, anti-Sca1 PE, anti-CD16/32 PerCP, anti-CD34 efluor450, anti-B220 FITC, anti-Gr1 FITC, anti-Ter119 FITC, anti-CD3 FITC, and anti-CD127 APC were purchased from eBioscience. The anti-CD11b PE, anti-CD11c APC, anti-Ly6C FITC, anti-CD117 PECy7 and anti-CD43 PerCP were purchased from BD Bioscience. After staining, cells were analyzed by flow cytometry on a FACS Canto. Data were analyzed with the FlowJo software.

Cells were isolated from the retina and choroid as described previously [62 (link)]. The samples were analyzed using FACSAria III (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, Ashland, OR, USA). Anti-F4/80-APC, anti-Ly6C-FITC, anti-Ly6G-APC-Cy7 (all from BioLegend, San Diego, CA, USA), and anti-CD11b-PE (eBioscience, San Diego, CA, USA) were used. Monocytes subsets were identified as CD11bhiF4/80hiLy6Chi/int/lo and can be subdivided into 3 subsets; classical, intermediate, and non-classical monocytes [63 (link)].