Flow cytometric analysis of murine immune cells

Mouse immune cells were stained for Live/Dead (L/D) (Invitrogen), CD45, CD3, CD4, CD8, IFN-γ, and TNF-α (Figure 3). To stain for the intracellular marker IFN-γ and TNF-α, samples were fixed in 1:3 fixation/permeabilization concentrate: diluent mixture (eBioscence) for 30 minutes and subsequently stained in permeabilization buffer (eBioscience). Fluorescence minus one (FMO) was used to control for data spread due to multiple fluorochromes and nonbinary expression of markers such as IFN-γ and TNF-α. Flow data were acquired using a FACSCelesta flow cytometer (BD) and analyzed using FlowJo (BD). Nonviable cells and doublets were excluded by forward versus side scatter gating, forward scatter height versus forward scatter area gating, and L/D staining.

Flow cytometric analysis of harvested tumor-bearing mouse brain with polymer-based vs systemic vs intracranial delivery of anti-PD-1. Mice were either not treated or given intracranial hydrogel (loaded with 200 ug anti-PD-1), intraperitoneal injection of anti-PD-1 (total dose 600 ug spaced over five days), cervical hydrogel (loaded with 600 ug of anti-PD-1), and inguinal hydrogel (loaded with 600 ug of anti-PD-1). (a) Flow cytometry plots demonstrating %parent populations of CD4+ and CD8+ IFN-γ expression in the brain of mice bearing GBM (n = 5 per arm). (b) Summary plots of IFN-γ activity in CD4+ and CD8+ cells based on treatment modality (n = 5 per arm). (c) Flow cytometry plots demonstrating %parent populations of CD4+ and CD8+ TNF-α expression in the brain of mice bearing GBM (n = 5 per arm). (d) Summary plots of TNF-α activity in CD4+ and CD8+ cells based on treatment modality (n = 5 per arm). (*** P < .001, ** P < .01, * P < .05)