Lab tek 2 8 chamber

Confocal microscopy was performed according to a modified version of a method described previously^{33 (link)}. Tumor sphere cells were seeded in a Lab Tek II 8-chamber (#155409; Thermo Scientific, Waltham, MA, USA) coated with Cell Tak (#354240; Corning, Corning, NY, USA). The sphere cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with anti-FDPS antibody (#ab153805; Abcam) and Alexa Fluor 488-conjugated anti-rabbit antibody (#A11008; Thermo Scientific). Nuclei were stained with DAPI (#268298; Calbiochem, San Diego, CA, USA). Images were obtained at ×20 at the Imaging Core (National Cancer Center) on a LSM510 META or LSM780 confocal microscope (Carl Zeiss, Jena, Germany). For determination of the type of cell death, TS cells were treated with various death-inducing reagents, stained with Hoechst (#H3570, Thermo, 10 μg/ml) and propidium iodide (#LS-02-100, Biobud, 0.3 μg/ml) and then imaged with the LSM780 confocal microscope. Z-stack orthogonal projection images were processed with ZEN 2012 analysis software (Carl Zeiss, Germany).

Confocal microscopy was performed as described previously.^[39] Briefly, cells were seeded in a Lab Tek II 8‐chamber (Thermo Scientific) and treated with or without Dox for 3 days. Just before imaging, Q‐annexin V (1 × 10⁻⁶m dye equivalent), PI (3 µg mL⁻¹), and DAPI (2 µg mL⁻¹) were added to the cell culture media. Confocal images (λ_ex 633 nm, λ_em 647–754 nm for Q‐annexin V; λ_ex 488 nm, λ_em 566–718 nm for PI; λ_ex 405 nm, λ_em 415–509 nm for DAPI) were obtained at the Microscopy Core Facility (National Cancer Center) on a LSM510 META (Carl Zeiss, Jena, Germany).