Brdu assay

Cells were washed with PBS (4 °C) and harvested by scratching. Subsequently, the cells were incubated with a phycoerythrin (PE)-labeled primary anti-NG2 antibody for 1 h at room temperature. Afterwards, the cells were washed in PBS and the MFI of 3000 cells was analyzed by a FACSLyrics flow cytometer (BD).
Cell proliferation was assessed by a BrdU-assay according to the manufacturer’s protocol (Thermo Scientific). Briefly, A1207 and U87 cells were incubated with BrdU. After 10 h, the cells were washed, fixed and permeabilized. Incorporated BrdU was detected by using a BrdU antibody. The MFI of 3000 cells was analyzed in the FL-1 and FL-2 channel by a FACSLyrics flow cytometer.

The proliferation of lung cancer cell was analyzed by Brdu assay (Thermofisher) as described by manufacturer. The co-culture of lung cancer cells and macrophages was conducted using Corning trans-well inserts (Sigma-Aldrich, St. Louis, MO, USA) as previously described [39 ].

Cells were washed in PBS and harvested by scratching. Subsequently, the cells were incubated with the phycoerythrin (PE)-labeled primary antibodies and the PE-labeled IgG control antibodies for 1 h at room temperature. Then, the cells were washed in PBS and the mean fluorescence intensity (MFI) of 5000 cells was analyzed in the FL-2 channel by a FACScan flow cytometer (Becton Dickinson, San Antonio, USA) using the CellQuest software (version 5.1).
Proliferative cells were detected by a BrdU-assay (Thermo Scientific Bremen, Germany) according to the manufacturer’s protocol. Briefly, pericytes were incubated with BrdU. Then, the cells were washed, fixed and permeabilized. BrdU was detected by using a BrdU-antibody provided by the manufacturer. The MFI of 5000 cells was analyzed in the FL-1 and FL-2 channel by a FACScan flow cytometer using the CellQuest software (version 5.1).