Brdu antibody

KG-1, Raw264.7 cells or hUCMSCs were detached by Accutase®. Then cell pellets were collected at 1200 rpm for 5 min and washed twice with PBS. Afterward, cells were suspended in 100 μL PBS and labeled with different macrophages markers iNOS (Abcam, Boston, USA) and CD163 (BioLegend, CA, USA). hUCMSCs were labeled with CD105 (BioLegend, CA, USA). For the cell proliferation experiment, cells incubated were treated with 10uM BrdU for an hour then stained with BrdU antibody (Thermo, Logan, UT). After incubation of the antibody, cells were washed with PBS three times and analyzed immediately by flow cytometry (Beckman, IL, USA).

Medulloblastoma cell line D458 was cultured in DMEM/F12 media with 10% FBS, 2 mM L-glutamine, and 1% Penicillin/Streptomycin at 37°C in an atmosphere of 5% CO₂.
Cell proliferation was measured by WST-1 or BrdU assays. For WST-1 assay, we used the cell proliferation reagent WST-1 (Millipore Sigma) according to its protocol. For BrdU assay, procedures were performed according to the protocols as described⁵. Briefly, we added the BrdU (10 μM) into medium, cultured the cells for 3 h at 37°C in an atmosphere of 5% CO₂, fixed cells with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. For BrdU staining, cells were treated with 1N HCl incubating 10 min on ice and neutralized by phosphate/citric acid buffer. BrdU antibody (Thermo Fisher Scientific, # MA3-071) was added into the cells and incubated overnight at 4°C. Mouse-anti BrdU (BD Bioscience, 1:500) antibody was used to label BrdU overnight at 4°C. DAPI counterstain was included in the final washes before the samples were mounted in Fluoromount G (SouthernBiotech) for microscopy. Cell images were quantified in a blinded manner. All immunofluorescence-labeled images were captured using a Nikon C2+ confocal microscope.