Anti cd11b apc

To detect mouse MDSC subsets, cell suspensions isolated from the spleens and kidneys were first incubated with Fc-blockeranti-CD16/32 antibody (dilution 1:20, Miltenyi Biotech) for 15 min, stained and pre-incubated with anti-CD11b-APC (1 μl/test) and anti-GR-1-PE (0.3 μl/test) for 30 min at 4°C in the dark. Cells were then washed with buffer to remove the excess stains and analyzed in a FACS (Becton Dickinson, San Diego, CA, USA). To detect TLR7 of MDSCs in pristane-treated mice, after stained with anti-CD11b-APC and anti-GR-1-PE, fixed and permeabilized by BD Fixation/Permeabilization Solution Kit (BD Pharmingen, USA), stained with anti-TLR7-FITC (Invitrogen, USA) for 30 min at 4°C.

Mesenteric and liver (celiac, portal) lymph nodes cells were isolated, washed, and counted. Cells were treated with FcR-block (Miltenyi Biotec) and stains were performed. Inflammatory monocytes: anti-CD11b-APC (Miltenyi Biotec, clone M1/70.15.11.5), anti-Ly6g-PacB (Biolegend, clone 18A), anti-F4/80-PE (eBioscience, clone BM8), anti-Ly6c-FITC (Novus Biologicals, clone HK1.4). T-cell hematopoiesis: anti-CD3e-APC (Miltenyi Biotec, clone 145-2C11), anti-TCRγδ-e450 (eBioscience, clone eBioGL3), anti-CD8a-PE (Miltenyi Biotec, clone 53-6.7), anti-CD4-FITC (Miltenyi Biotec, clone GK1.5). Dead cells were excluded via propidium iodide viability dye (Miltenyi Biotec). Data was acquired by the MACSQuant System (Miltenyi Biotec). Analyses were performed via FlowJo VX software (TreeStar).

To isolate microglia for WB analysis, TBI‐ or sham‐treated wild‐type mice were anesthetized, and the brain was dissected. Then the injured‐ or sham‐treated cortex was collected and digested using 0.25% trypsin. After being filtered with a 75 μm cell strainer, it was resuspended into 35% Percoll and centrifugated at 400 g to remove myelin as reported.
²⁰ (link) Then cell pellets were resuspended in PBS and labeled with anti‐CD11b‐APC (Miltenyi, 1:50) and anti‐CD45‐FITC (BD, 1:100). CD11b and CD45 were used for distinguishing resident microglia (CD11b⁺, CD45^low), activated microglia (CD11b⁺, CD45^intermediate), and infiltrating macrophages (CD11b⁺, CD45^high) as reported.
³¹ (link) Gates were established using antibody isotype controls and fluorescence minus‐one controls. Samples were acquired by Beckman MoFlo Astrios Cell Sorter and analyzed with Flowjo 10.0 software. Only CD11b⁺, CD45^low, and CD11b⁺, CD45^intermediate cells were acquired, which indicated the microglia in the cortex specifically. Samples were then denatured with an SDS‐PAGE loading buffer and subjected to WB detection.

The saliva samples were diluted with phosphate-buffered saline (PBS) (1:10) and centrifuged at 1000 rcf for 10 min. The pellet obtained was then further subjected to antibody staining and flow cytometry analysis. Briefly, the cells were resuspended in PBS and labeled with anti-CD191-PE (1:100 dilution), anti-CD11b-APC (1:100 dilution), anti-CD192-APC (1:100 dilution), anti-CD185-PE (1:100 dilution), and anti-CD195-FITC (1:100 dilution) (Miltenyi Biotec, Bergisch Gladbach, Germany). After incubation for 30 min at room temperature, the cells were acquired on a flow cytometer (Attune NxT, Thermo Fisher Scientific, Waltham, MA, USA). A total of 5000 cell events were acquired in each sample. The median fluorescence intensities were calculated and compared.