Pe anti mouse cd11c antibody

Adipocyte adipose tissue obtained from epididymis fat cells was collected after extensive trituration in phosphate-buffered saline solution pre-chilled to stop cell growth, and an even suspension of a single cell was pipetted. In aliquots of 1.0 × 10⁶ cells, ten microliters of the cell suspension was removed and diluted 1000-fold to get a trypan blue stain, and counted under a microscope. To a blend of 1.25 μL PE anti-mouse CD11c antibody (117307, Biolegend, San Diego, CA USA), 2 μL FITC anti-mouse F4/80 recombinant antibody (157309, Biolegend, San Diego, CA USA), and 2.5 μL APC anti-mouse CD206 antibody (141707, Biolegend, San Diego, CA USA), fluorescently labeled CD45 antibody was added (CD45 anti-perCP antibody in the form of 103129 from Biolegend, San Diego, USA) and a 30 minute incubation at 4 °C in the dark was conducted. Centrifugation was performed after adding 2 mL of diluted buffer to the cells, and the supernatant was discarded after washing the cells. We resuspended the cells in Flow Cytometry Staining Buffer (500 μL) and then detected them with the FACS system. A CD45⁺F4/80⁺CD206⁺ macrophage was identified as M2 and a CD45⁺F4/80⁺CD11c⁺ macrophage as M1.

Transfected mice were euthanized and their spleens were filtered through a metal cell strainer. Single cells were resuspended in PBS and dendritic cells were labeled using PE anti-mouse CD11c Antibody (Biolegend). Cells were then analyzed using BD FACSCalibur. PE-positive cells were gated and expression of GFP was assessed among the gated cells.

Cells were cultured for 24 h after seeded on glass coverslips and then 2.5% bovine serum albumin was used as blocking reagent for 1 h. After that the cells were conjugated with PE anti-mouse CD11c antibody (Biolegend, San Diego, CA, USA) or PE anti-mouse CD206 antibody (Biolegend) at 25 °C for 1 h. The samples on the coverslips were gently mounted with 10 μL DAPI mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). The coverslips were sealed with the nail oil and images captured with Leica TCS SP5 confocal microscope.