10 mm brdu

Calu-3 or N87 cells (100,000/well) were seeded into 6-well plates and incubated overnight. Antibodies or DVD-Ig proteins were added at 100 nM and cells were treated for two days. Cells were pulse-labeled with 10 mM BrdU (BD Biosciences, CA) for 30 minutes at 37°C and then harvested and stained with BrdU-FITC (BD Biosciences, CA) according to manufacturer’s protocol.

GBM cells were cultured on type I collagen for 24 h and treated as indicated. Before harvesting, cells were incubated with 10 mM BrdU (BD Biosciences) for 10 min and then prepared for cell cycle measurement. Cells were harvested using 1x trypsin/EDTA and fixed in icecold 80% ethanol until use. Cells were further prepared for analysis by incubation with 0.01 % RNase A, 2 N HCl (all Sigma-Aldrich) and 0.5% Triton-X-100/PBS (Carl Roth GmbH). Subsequently, mouse anti-BrdU antibodies and propidium iodide (Sigma-Aldrich) was added for the detection of incorporated BrdU and total DNA content. Cell cycle distribution was determined using an FACS Calibur (BD Biosciences) and analyzed using the FlowJo Software (Flowjo LLC).