Calcein am pi cell live dead assay kit

After CFSE dye staining, Cal27 cells were observed for their behavior by an inverted microscope (Zeiss VERT1, United States) and LSCM (Olympus Fluoview FV1200, Tokyo, Japan). Cells encapsulated in microtubes were stained with Calcein-AM/PI Cell Live/Dead Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) to investigate the viability of cells.

In order to ensure the availability of essential nutrients for cellular growth, the microfluidic chip was continuously perfused with nutrient media using a syringe pump (LSP02-2A, Longerpump, China). The culture medium in the syringes was separately supplemented with PBS, blank MΦ, nab-PTX, and nab-PTX/MΦ, divided into four groups for perfusion to the microfluidic chip. Following a 48-h incubation period, the viability of 3D multicellular tumor spheroids was assayed using Calcein-AM/PI Cell Live/Dead Assay Kit (Beyotime). Viable cells were identified using Calcein-AM (green), while non-viable cells selectively took up PI (red).
U87-EGFP cells, expressing green fluorescent protein (EGFP), were also employed to generate GBM spheroids for prompt and distinct assessment of the anti-GBM effects. The fluorescence emitted by these cells enables visualization and tracking of their behavior within the tumor microenvironment. By monitoring the fluorescence intensity or distribution of U87-EGFP cells over time, we can evaluate various aspects of tumor growth, invasion, and response to treatment. Following 48 h of diverse interventions, the U87-EGFP spheroids were observed using LSCM.