Pe anti human cd11b

Cell cycle analysis was performed with a cell cycle detection kit (#KGA511; KeyGen, Nanjing, China) according to the manufacturer's instructions. The treated cells were fixed with 75% ethanol and then stained with PI for flow cytometry analysis.
To detect the phenotype of PBMC‐DMs, cells were incubated with indicated CMs for 48 h after differentiation. The phenotype of the macrophages was analyzed by flow cytometry, independently, followed by staining with APC anti‐human CD209 (#330107), FITC anti‐human CD80 (#305205), APC anti‐human CCR7 (#353213), PE anti‐human CD11b (#301305), FITC anti‐human CD36 (336203), Alex Fluor^@700 anti‐human CD16 (#302026), PerCP anti‐human CD14 (#325632), PE/Cyanine 7 anti‐human CD45 (#368532), Brilliant Violet 421 anti‐human HLA‐DR (#307635), and APC anti‐human Lineage cocktail (CD3, CD19, CD20, CD56) (#363601), which were all purchased from BioLegend (San Diego, CA, USA).

Using the freshly isolated neutrophils, we performed staining to detect neutrophil surface markers and apoptotic cells. The antibody clones (Supplementary Table S1) used for flow cytometric analysis were: Annexin V-APC (Vazyme), propidium iodide (PI; Vazyme), anti-human CD11b-PE (Biolegend), and anti-human CD15-PE-Cy7 (Biolegend). The mixture of these staining antibodies was directly added to suspensions containing 5 × 10⁵ freshly isolated neutrophils and kept for 20 min at room temperature. After this incubation, we washed the cells with PBS and prepared them for analysis.

BM, spleen (SPL), and peripheral blood (PB) were obtained from mice. Single-cell suspensions were prepared from each tissue following standard procedures. Erythrocytes were hemolyzed using BD Pharm Lyse buffer (BD Biosciences). Leukocytes were stained with fluorochrome-conjugated human antibody/mouse antibody for 20 min at room temperature in FACS buffer. After washing the labeled cells in 1x PBS, the cells were re-suspended in FACS buffer. Fluorescent signals were detected with a FACSVerse^TM flow cytometer, and data were analyzed using FlowJo software (BD Biosciences). The following human antibodies were used to measure human engrafted hematopoietic cells and mouse hematopoietic cells: anti-human CD45-APC and anti-human CD66b-FITC were purchased from Beckman Coulter. Anti-mouse CD45-FITC, anti-human CD33-FITC, anti-human CD14-PE, anti-human CD11b-PE, anti-human CD41-PE, anti-human CD235a-FITC, anti-human CD3-FITC, anti-human CD19-APC were purchased from BioLegend.

CD14⁺ cells were isolated from human PBMCs using anti-human biotin CD14⁺ antibody (BioLegend; Cat. 325624) and cultured in macrophage differentiation media (RPMI1640 + 20% FBS + 1% P/S + 20 ng/mL M-CSF) for 6 days and 30,000 cells seeded overnight in a new plate. On day 7, 60,000 RAJI-GFP cells (target cells) were preincubated with STI-6643 or rituximab for 30 minutes at 37°C followed by addition to the plate containing the macrophages and incubated for 1 hour. Macrophages were labelled with anti-human CD11b PE (BioLegend; Cat. 982606) for 15 minutes prior the end of the incubation. Phagocytosis of target cells were measured as percent CD11b⁺GFP+ macrophages on Attune flow cytometer.
Alternately, Cell Proliferation Dye eFluor™ 670 (eBioscience; Cat. 65-0840-85) was used to label MDA-MB-231-Fluc target cells and RAJI-GFP were used without prior labelling. MDA-MB-231 cells were incubated for 2 h at 37°C prior to adding anti-CD47 (STI-6643 or Hu5F9) or anti-PD-L1 mAbs for 30-minutes. Then, PBMCs were added and incubated for 90 minutes at 37°C. After incubation, cells were resuspended in FACS buffer containing anti-human CD14-PE (BioLegend, Cat. 325606) antibody and incubated for 15 minutes at 4°C, washed, fixed, and analyzed by flow cytometry as above.

Human macrophages were isolated as described previously [14 (link)]. In brief, meninges were removed and the tissue was mechanically dissociated using a glass tissue homogenizer. A cell suspension was obtained via filtering through 300-µm and 106-µm sieves. Myelin was removed by a 24% Percoll gradient (Fisher Scientific, 17-0891-01) in 10 × HBSS (Gibco, 14180-046) and phosphate buffered saline (PBS) density gradient centrifugation and followed by a second centrifugation step containing a 60% and 30% Percoll layer with PBS on top. The interphase between the Percoll layers was collected and contained the immune cells. Fc receptors were blocked with human Fc receptor-binding inhibitor (eBioscience, 14-9161-73). For FACS, cells were incubated for 20 min with anti-human CD11B-PE (BioLegend, 301306) and anti-human CD45-FITC (BioLegend, 304006) and washed with HBSS without phenol red (Thermofisher Scientific, 14175-053). The cells were passed through a 35-μm nylon mesh, collected in round bottom tubes (Corning 352235), stained with DAPI (Biolegend, 422801) and DRAQ5 (Thermofisher Scientific, 62251) and sorted using a Beckman Coulter MoFloAstrios, Beckman Coulter MoFloXDP or Sony SH800S cell sorter. Human macrophages were sorted as DAPI^negDRAQ5^posCD11B^posCD45^pos (Additional file 2: Fig S2a).