Vybrant dye

Cells were harvested after trypsinization and washed with PBS. To separate G1-phase cells from G2/M-phase cells, cells were incubated at 37 °C with Vybrant dye (V35004; Life Technologies) for 30 min and were then sorted based on their DNA content using a FACSAria III (Becton Dickinson). Sorted cells were incubated at 37 °C in 5% CO₂ overnight before treatment with DMSO or KU + Y. To separate cells with different senescence states, the forward scatter (FSC) and FITC channel intensities were obtained as the measures of cell size and lipofuscin content, respectively, and cells were then sorted based on these intensities.

To test cellular dyes, the cells are plated on collagen-coated tissue culture plastic, fluorescently labeled with a range of concentrations of various CellTracker dyes (Life technologies) and Vybrant dyes (Life Technologies) according to the manufacturer’s suggested protocol and maintained in respective serum-free media. Cells are also tested in varying media compositions to determine optimal viability. Tested media compositions include basal astrocyte medium alone (Sciencell) or supplemented with 1% B27 without vitamin A and 0.5% N2 and/or 0.01% FGF and 0.1% EGF. For all tests, the growth of labeled cells is measured after 18, 48, and/or 72 h using the CCK-8 cell proliferation and cytotoxicity (Dojindo) kits according to manufacturers’ suggested protocols. After 72 h, cells are also assessed for viability using Live and Dead ReadyProbes Reagents (Life technologies), imaged using wide-field microscopy with EVOS FL Auto (Life technologies), and quantified using ImageJ (National Institutes of Health). Each CellTracker or Vybrant dye test was performed similarly for each glial cell type (Supplementary Fig. 3).

Experiments are carried out in tissue culture inserts with 8 µm pore size (Sigma Aldrich CLS3374). Cells are fluorescently labeled with CellTracker dyes (Life technologies) and Vybrant dyes (Life Technologies) according to the manufacturer's suggested protocol. GBM cells (5.0 × 10⁵), astrocytes (8.0 × 10⁴), and microglia (8.0 × 10⁴) are seeded in 75 µL gel comprising (0.2% hyaluronan; ESI Bio) and 0.12% rat tail collagen I (Corning) according to cell ratios quantified from human sections. The gels are plated within 96-well tissue culture inserts with 8 µm pores (Corning) and cross-linked at 37 °C in a humidified incubator containing 5% CO₂ and 21% O₂. After 3 h, serum-free medium (Astrocyte Basal; Sciencell, with 1% B27, 0.5% N₂) is added to the top and bottom of each tissue culture insert. For static conditions, the medium level is consistent inside and outside the insert, while the medium on top of the gel is greater in flow conditions. This equates to 25 µL of medium under and 125 µL of medium on top for flow, and the reverse in static. Hydrogel stiffness was previously optimized to recreate mechanical properties of the brain^{22 (link)}.