Apc conjugated anti brdu antibody

RAW264.7 cells were seeded at 0.2 x 10⁶ cells/well in 6-well plates, cultured in DMEM with 10% FBS at 37^°C for 12 h. After 12 h, cells were infected with Mtb strains at MOI 5 and incubated for 12, 24, 36 and 48 h. Prior to harvesting, the cells were pulse labelled with bromodeoxyuridine (BrdU) for 45 min. Uninfected cells and unlabelled BrdU cells were used as negative controls. The harvested cells were stained with the live/dead stain (Ex ~633 nm/ Em ~780 nm, Invitrogen), washed and fixed in ice cold 70% ethanol for 12 h. After washing with PBS, the cells were treated with 2N HCl for 20 min to expose BrdU labeled DNA and the acidic pH was neutralized with 0.1 M sodium borate buffer, pH 9.0. The cells were then permeabilized with 1X permeabilization buffer (BD Biosciences) containing 0.3% Triton X-100 to ensure nuclear membrane permeabilization and washed once with PBS. Cells were labelled with APC-conjugated anti-BrdU antibody (Biolegend) for 1 h on ice. After washing by centrifugation, the cells were resuspended in 0.2 ml of propidium iodide solution (Sigma), and allowed to incubate for 30 min on ice. Cell cycle analysis was performed by data acquisition with Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo Vx.0.7. Two independent experiments were performed in triplicate.

Cells were treated with 10 µM BrdU (BioLegend) for 2 h. After washing twice with PBS, the cells were fixed in ice-cold 70% ethanol for 30 min at 4 °C. The cells were resuspended in 1 ml freshly prepared 2 M HCl/0.5% Triton X-100/PBS and incubated for 20 min at room temperature. After washing twice with PBS, the cells were treated with 1 ml 0.1 M sodium tetraborate/0.5% Tween/PBS for 10 min at room temperature. The cells were washed with PBS and incubated in 100 µl antibody solution (0.4 µl APC-conjugated anti-BrdU antibody, BioLegend in PBS with 5% FBS) for 30 min at room temperature. After washing, the cells were treated with 50 µl RNase A (10 µg/ml) for 30–60 min. Finally, cells were stained with 3.3 µg/ml DAPI for 10 min. Flow cytometry was performed with a FACS Canto II (BD BioSciences) and data were analyzed by FlowJo V9.