Anti mouse cd16 cd32 mab

Macrophages were treated with 25 μg/mL of either MWCNTs or MWCNT–OVA complex for 6 hours and washed and incubated up to 5 days in complete medium supplemented with 5 ng/mL M-CSF. At day 1, day 3, and day 5, the supernatants were collected for the measurement of tumor necrosis factor-α (TNFα) and interleukin (IL)-6 production using enzyme-linked immunosorbent assay according to the manufacturer’s protocol (R&D Systems, Inc.). For MHCII expression, macrophages were detached using 0.025% trypsin and resuspended in staining buffer (Thermo Fisher Scientific, Waltham, MA, USA). Rat monoclonal PE/Cy5-conjugated anti-MHCII (PE/Cy5) antibody (Abcam, Cambridge, UK) was used to detect MHCII expression on macrophages using flow cytometry (Accuri™ C6 Flow Cytometer; BD Biosciences, San Jose, CA, USA). Prior to labeling MHCII molecules, macrophages were pre-incubated with purified anti-mouse CD16/CD32 mAb (BD Pharmingen; BD Biosciences, San Jose, CA, USA) to reduce Fc-receptor-mediated nonspecific binding. OVA was used as a positive control. The experiments were conducted independently on a minimum of three different batches of BMDMs.

To minimize nonspecific bindings of mAbs, single-cell suspensions were preincubated with anti-mouse CD16/CD32 mAb (BD Pharmingen, San Jose, CA), then washed and incubated with the indicated mAb conjugates for 15 min on ice. The stained cells were resuspended in staining buffer containing 1 μg/ml PI for the elimination of dead cells. Flow cytometry was performed using an Attune Acoustic Focusing Cytometer (Applied Biosystems, Foster City, CA), and all data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR). The absolute number of cells infiltrating into the kidney was quantified by flow cytometry using CountBright absolute counting beads (Life Technologies, Waltham, MA) following the instructions from the manufacturer. For cell sorting of renal Ly6G⁺ cells, single-cell suspensions were stained with FITC-Ly6G, PE-CD11b, PI and PE-Cy7-F4/80. Ly6G⁺ cells among the CD11b⁺/F4/80^low population were then sorted using an S3 Cell Sorter (Bio-Rad, Hercules, CA). Sorted cells were stained with May-Grünwald -Giemsa, and cell morphology was captured under light microscopy (BX51 microscope; Olympus, Tokyo, Japan).