Anti gr 1 apc

When the lung-infiltrating cells were analyzed, anti-CD45-APC-Cy7 (BioLegend) were intravenously injected (3 μg/mouse) before euthanasia to exclude the blood-circulating immune cells. Cells from lung or spinal cord tissues were prepared by enzymatic digestion with 2.5 mg/ml collagenase D (Sigma-Aldrich) and 0.1 mg/ml DNase I (Sigma-Aldrich) for 30 min at 37°C. For neutrophils, monocytes, macrophages, and CD4⁺ T cell staining, the cells or tissues were stained with anti-Ly6c-AF488 (BioLegend), anti-CD45.2-PE (BioLegend), anti-CD11c-PE (BioLegend), anti-CD11b-PerCP (BioLegend), anti-Gr-1-APC (eBioscience), anti-CD4-APC (TONBO), and anti-F4/80-BV421 (BioLegend).

For staining the membrane antigens of BM cells, single-cell suspension was prepared. After treatment with exosomes, 1 × 10⁶ cells were resuspended in PBS with 5% BSA and stained with PE-Cy7-conjugated antibodies to CD11b (anti-CD11b-PE-Cy7, 101216, BioLegend, San Diego, CA, USA) and with 3H2, an anti-idiotype antibody against 5T33MM cells [54 (link)], followed by an APC-conjugated rat anti mouse IgG1 (550874, BD Biosciences, San Jose, CA, USA). For staining the membrane markers of MDSCs, the cells were stained with anti-Gr-1-APC (17–5931, eBioscience) and anti-CD11b-PE-Cy7 or with anti-Ly6G-PE-Cy7 (127618, BioLegend) and anti-Ly6C-APC (560595, BD Biosciences). Staining with Annexin V-fluorescein isothiocyanate (Annexin V-FITC, 556419, BD Biosciences) was used to determine the apoptotic cells. For staining intracellular p-Stat3 and p-Stat1 in MDSCs, cells were first fixed with formaldehyde and methanol, followed by staining with anti-Gr-1-APC, anti-CD11b-PE-Cy7, anti-p-Stat3-Alexa Fluor 488 (557814, BD Biosciences), and anti-p-Stat1-PE (612564, BD Biosciences). The percentage of a certain population and mean fluorescence intensity was evaluated using a FACSCanto flow cytometer (BD Biosciences) and Flowjo software (TreeStar, Ashland, OR, USA).