Ln229

Human GBM cell lines U87MG and LN229 and the mouse GBM cell line GL261 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The human monocyte leukemia cell line (THP-1) was a kind gift from Professor Yuan Guo, Department of General Medicine, Shandong University. U87MG, LN229, GL261, and THP1 were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific). THP-1 cells were treated with 200 nM phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich; St. Louis, MO, USA) for 24 h to allow for differentiation into macrophages in six-well plates. All cells were maintained at 37°C in a cell incubator containing 5% CO₂.

The human glioma cell lines U87, U251, Ln229, U373 and U118 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). A primary culture designated GBM1 (GP1) was established in February 2016 from the tumor cells of a patient with a left frontal glioblastoma, and a culture designated GBM2 (GP2) was established in March 2016 from tumor cells taken from a patient with a left tempus glioblastoma. Tumor tissue was collected from a patient who granted written informed consent. The Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University approved the study protocol.
Tissue was obtained from regions comprising viable tumor cells. Within 2 h after acquisition, the tissue samples were dissociated into single-cell suspensions, washed with Hanks solution (Solarbio, Beijing, China) to remove red blood cells, and the number of cells was counted. All cell lines were cultured at 37 °C in 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone Laboratories, Utah, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, CA, USA). Hypoxia was achieved using a hypoxia incubator HEPA filter (Thermo Fisher Scientific, Boston, USA) flushed with 2% O₂, 5% CO₂ and 93% N₂.

The GBM cell lines U87, U251, LN229, and T98 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin/mL, and 100 ng of streptomycin/mL. Two primary human GBM cells (pGBM1 and pGBM2) were obtained from primary human GBM samples as described previously [25 (link)]. For cell culture, all cells were incubated in 5% CO₂ at 37° C.

The human glioma cell lines U251, LN229, A172, U87, U118, and H4 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). Normal human astrocytes (NHAs) were obtained from Lonza (Basel, Switzerland) and cultured as recommended by the manufacturer. A primary culture designated patient primary GBM cells (PG1) lines was established in April 2017 from the tumor cells of a patient with a right frontal glioblastoma who granted written informed consent. The Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University approved the study protocol.
Tissue was obtained from regions comprising viable tumor cells. Within 2 h after tumor tissue collection, tissue samples were dissociated into single cell suspensions, and washed with Hanks’ solution (Solarbio, Beijing, China) to remove red blood cells. The number of cells was then counted. Primary cultures were maintained in serum, and all cell lines were maintained in Dulbecco’s modified Eagle’s medium with sodium pyruvate supplemented with 10% fetal bovine serum with high glucose and antibiotics (100 U of penicillin/mL and 100 ng of streptomycin/mL). All cell lines were grown at 37 °C in a humidified 5% CO₂ atmosphere.

The human astrocytes (HA) cell line was obtained from ScienCell Research Laboratories. Human glioma cell lines such as LN229 and U251 were purchased from the Chinese Academy of Sciences Cell Bank. The cell lines were cultured in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were maintained in a 5% CO₂ humidified incubator at 37°C.