Flow Cytometry Protocol for Cell Death Analysis in Glioblastoma Organoids: A Technical Note v1

Here, we present a protocol to quantify cell death in glioblastoma organoids (GBOs) using flow cytometry. Single-cell suspensions are generated through combined enzymatic and mechanical dissociation, followed by permeabilization and nuclear staining with propidium iodide or Hoechst 33258 to detect DNA fragmentation. Following treatment with temozolomide (TMZ) and lomustine (CCNU) for up to 288 hours, flow cytometric analysis revealed cell death rates of up to 63%, with CCNU demonstrating greater efficacy than TMZ. The extent of cell death varied between GBO populations but was consistent across replicates derived from the same GBO population. The protocol is scalable, cost-effective, and suitable for high-throughput applications in translational cancer research.