Cd11b pe antibody

Mouse whole blood was collected with 1.5% EDTA-2Na used as the anticoagulation agent, and red blood cells were eliminated by a lysing buffer (BD Pharmingen). Leukocyte cells and BMDCs of normal control mice or OSC-19 tumour-bearing mice were stained with CD11b-PE antibody (BD Pharmingen). For IL-13Rα2 and CD11b double staining, OSC-19 tumours grown at non-IR sites or pre-IR sites in nude mice were minced and enzymatically digested with 3 mg/ml Type VI collagenase and 2 U/ml hyaluronidase (SIGMA). They were further treated with 0.1 mg/ml DNase I (Wako) and 0.09% NH₄Cl and strained through a 40 μm cell strainer. Single cells were incubated with IL-13Rα2-biotin (R&D systems) and CD11b-PE antibodies (BD Pharmingen), followed by Streptavidin-APC antibody (eBioscience). All samples were pre-incubated with Fc-block (BioLegend) prior to staining. Analysis was performed using MoFlo® Astrios (Beckman Coulter) and Summit Software ver. 4.3 (Beckman Coulter).

For the isolation of myeloid-derived suppressor cells (MDSCs), 1×10⁵ TC-1 cells were injected subcutaneously into wild type C57BL/6 mice. 20 days later, splenocytes were collected from naïve (control) or tumor-bearing mice and prepared for flow cytometry analysis to measure CD11b and Gr-1 positive cells. Splenocytes were stained with FITC-conjugated Gr-1 antibody (BD Pharmingen, San Diego, CA) and PE-CD11b antibody. MDSCs were purified by magnetic cell sorting using the mouse CD11b MicroBeads according to the manufacturer's instructions (MACS; Miltenyi Biotec, Auburn, CA). The purity of CD11b⁺ cells obtained by this method was 90–95% and 90% of CD11b⁺ cells were Gr-1⁺ as determined by flow cytometry. For the detection of MDSCs in tumor tissue, TC-1 tumor tissues obtained from tumor-bearing mice were cut into fragments in phosphate-buffered saline (PBS), washed twice with PBS, and then digested with 500 U/ml of Dispase (Godo Shusei, Co., Ltd. Tokyo) at 37°C for 20 min. The supernatants of the digestion were discarded and the remaining fragments were suspended into 5 ml of PBS. The cell suspensions were passed through a stainless wire sieve (100 mesh), washed twice with 20 ml of PBS and centrifuged for 5 min at 15O×g. Pelleted cells were resuspended in PBS and stained with FITC-conjugated Gr-1 antibody (BD Pharmingen, San Diego, CA) and PE-CD11b antibody.

Fresh tumor samples were collected in cold HBSS (Hank's Balanced Sodium Solution without Ca²⁺ and Mg²⁺) with antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin) and processed within 24 h. The tissue was mechanically and enzymatically dissociated with the gentle MACS™Dissociator and Neural Tissue Dissociation Kit (MiltenyiBiotec, Cologne, Germany). Cells were labeled with CD11b MicroBeads, loaded onto MACS Columns placed in the magnetic field of MACS Separator. CD11b⁺ cells were eluted as a positive fraction, stained with CD11b-PE antibody (BD Pharmingen, San Diego California) and checked for purity using flow cytometry (FACSCalibur, BD Bioscences, NJ, USA) and CellQuest software. Total RNA from sorted CD11b⁺ cells was isolated with RNeasy Mini Kit (Qiagen, Hilden, Germany). Relative quantification of gene expression was determined using the comparative CT method.