Prior studies demonstrated a proclivity of gliomas to acidify heterogeneously but, generally, with increased proton concentration toward the tumor core. the hyperpolarization-dependent G1-to-S phase cell cycle checkpoint, thereby inhibiting cell division. In this way, pHe directly determines the proliferative state of glioma cells. mouse pups, passaged once, and then plated on coverslips. All cells were incubated in variants of DMEM-Ham’s F-12 medium (DMEM/F-12; Invitrogen) with 7% FBS. Tumor spheroid formation. U251-MG human glioma cells were plated in 200 l of DMEM/F-12 with 7% FBS at a starting concentration of 5 103C1.5 104 cells/well into agarose-coated 96-well plates. The plates were coated with autoclaved 1.5% (wt/vol) agarose-containing DMEM/F-12 (50 l/well) and cooled until the agarose hardened. Initial spheroid formation occurred 2 days after the cells were plated; the spheroids were grown in an incubator in bicarbonate-buffered DMEM/F-12 at 37C and 10% CO2 for 1 wk. After 1 wk, spheroid diameter was 100C500 m. Paraffin embedding. Spheroids >1 wk old were collected and fixed with 4% paraformaldehyde in PBS, dehydrated in ethanol, and embedded in HistoGel blocks (Thermo Scientific) using cryomolds. HistoGel was processed to paraffin and Salmefamol then embedded in a paraffin block. Sections (7 m) were cut using a Leica microtome and placed on positively charged glass slides (catalog no. 12-550-17, Fisher Scientific). Immunocytochemistry. The paraffin sections of tumor spheroid were deparaffinized using WNT4 CitriSolv (catalog no. 22-143-975, Fisher Scientific), rehydrated, and washed with PBS. The spheroids were then blocked and permeabilized in PBS containing 0.3% Triton X-100 and 10% normal goat serum and then stained overnight at 4C with rabbit anti-Ki67 antibody (1:1,000 dilution; catalog no. 15580, Abcam) in a 1:2 dilution of blocking buffer (BB) in PBS. After the slides were washed in PBS, they were stained with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:500 dilution; catalog no. A-11008, Invitrogen) in the diluted BB for 2 h at room temperature, washed, and incubated for 5 min in PBS with 1 mg/ml 4,6-diamidino-2-phenylindole (DAPI, 1:1,000 dilution; catalog no. 62248, Thermo Scientific) and then mounted with Aqua-Poly/Mount (catalog no. 18606, Polysciences) and a cover glass. Images were acquired Salmefamol using a Zeiss Axiovert 200M microscope with a 20 air objective and Axiovision release 4.6 software. Ki67 staining was visualized using a FITC filter set, while Salmefamol DAPI staining was visualized using a DAPI filter set. pH recordings of spheroids. Spheroids >1 wk old were preincubated in sulfate- and phosphate-free (SPF) pH 6.0, 7.4, and 8.8 baths (for composition, see stacks of 5-m section thickness through the spheroid with Olympus Fluoview ASW 3.1 and analyzed using WCIF ImageJ. For the pHe and 3 for all experiments. RESULTS Glioma cells organically evolve gradients of pHe and cell proliferation in vitro. Prior studies demonstrated a proclivity of gliomas to acidify heterogeneously but, generally, with increased proton concentration toward the tumor core. The cause of this acidification is multifaceted and includes heterogeneous expression of acid extruders, poor access to nutrients, and a mix of cell populations (18). We wondered if tumor acidification could organically evolve from a clonal population of cells and in the presence of ample nutrients. Previous studies demonstrated a pHe gradient in rat gliomas in vivo (9, 10) and in glioma spheroids in vitro (1). To gain sufficient pHe resolution, we elected to use the cell-impermeant ratiometric pH indicator dye SNARF-5F (20 M) to analyze spheroid pHe gradients. U251 human glioma cells, which quickly aggregate, were grown to form spheroids with 104 cells and analyzed via confocal microscopy for pHe differences. We compared pHe gradients from spheroids bathed for Salmefamol 2 h in acidic, neutral, and alkaline pH-buffered media to determine = 3 each). Consequently, the proton concentration gradient was significantly steeper in the alkaline pH media solutions, where maximally there was an order-of-magnitude change in proton concentration every 7 m into the spheroid core (Fig. 1sections are shown at 20 magnification through the middle of the spheroids. and = 3) and maximum gradient of acidification (1-way ANOVA with Tukey-Kramer post test, = 3), respectively. *< 0.05; **< 0.01. We further hypothesized that this Salmefamol acidification inversely corresponded to tumor cell proliferation. Paraffin sections of 1-wk-old tumor spheroids were immunostained for Ki67,.