Accutase

T98G, U87MG, TS603 and 13.0302 cells were cultured in neurobasal medium (Stemcell technologies, 5751) containing 10% proliferation supplement (Stemcell technologies, 5751), 20ng/mL H-EGF (Stemcell technologies, 78006), 10ng/mL H-bFGF (Stemcell technologies, 78003) and 2μg/mL Heparin (Stemcell technologies, 7980). Once neurospheres were formed, they were dissociated by Accutase (VWR, 490007-741) into single cells. Single cells were seeded into 12-well plates (5,000 cells/well) and treated with control medium or THIO at 5 μM for 12 days. Phase contrast images of neurospheres were acquired by ECHO Revolve Microscope at day 3, 6, 9 and 12. At day 12, neurospheres were stained using Live Dead Cell Viability Assay Kit (Sigma-Aldrich, CBA415) and imaged by ECHO Revolve Microscope.

T98G, U87MG, TS-603, and 13.0302 cells were cultured in neurobasal medium (Stemcell Technologies, 5751) containing 10% proliferation supplement (Stemcell Technologies, 5751), 20 ng/mL H-EGF (Stemcell Technologies, 78006), 10 ng/mL H-bFGF (Stemcell Technologies, 78003), and 2 μg/mL Heparin (Stemcell Technologies, 7980). Once neurospheres were formed, they were dissociated by Accutase (VWR, 490007–741) into single cells. Single cells were seeded into 12-well plates (5,000 cells/well) and treated with control medium or Gamitrinib at 1, 2.5, and 5 μmol/L for 12 days. Phase contrast images of neurospheres were acquired by ECHO Revolve Microscope at days 3, 6, 9, and 12. At day 12, neurospheres were stained using Live Dead Cell Viability Assay Kit (Sigma-Aldrich, CBA415) and imaged by ECHO Revolve Microscope. Two independent experiments were performed.

For antibody staining, 200,000 cells were seeded in 6-cm dishes overnight. Cells were detached from plates with Accutase (EMD Millipore cat #SCR005) at 37^oC for 5 minutes, washed in PBS and incubated with anti-CCR2-PE diluted 1:50, (R&D Systems, cat# FAB151P) for 1 hour. For cell cycle studies, 200,000 cells were seeded in 6-cm dishes overnight. Cells were treated with or without 2mM Thymidine (Sigma, cat # T1895–1G) for 16 hours. The cells were washed 3 times with serum free media (BT-20: EMEM, HCC1937: RPMI, and MCF10CA1d: DMEM). Growth media was added for 2 hours for MCF10CA1d cells and 10 hours for BT-20 and HCC1937 cells. Cells were incubated with 2mM Thymidine for 16 hours, washed with serum free media and incubated with growth media, with or without 100 ng/ml CCL2. Cells were detached with Accutase and fixed with 70% ethanol at −20ᵒC. Samples were incubated in 500 μL of PBS/0.1% Triton X-100/2mg/ml RNase A (VWR, cat# 97064–064) and 200 μg/ml propidium iodide (Invitrogen, cat# P3566) for 15 minutes at 37ᵒC.
For ALDH activity assay, cells were seeded in 6 well plates (200,000/well), serum starved for 24 hours and incubated in serum free medium with or without 100 ng/ml CCL2 for 24 hours. Cells were subject to AldeRed™ Assay (EMT Millipore, cat #SCR150) according to commercial protocol. All cells were analyzed using a BD LSRII Flow Cytometer.