35 mm glass bottom culture dishes

Confluent HCT-116 cells were passaged onto 35 mm glass bottom culture dishes (Greiner bio one; cat. no. 627861), and allowed to grow until 70% confluence overnight. Cells were treated with bis-ANS or sodium arsenite dissolved in culture medium, and before imaging, cells were washed three times with Dulbecco’s phosphate-buffered saline (Gibco; cat. no. 14190-144), then imaged in Fluorobrite™ Dulbecco’s modified eagle medium (Gibco; cat. No. A18967-01) cell culture medium. In order to visualize the cell nuclei, the cell permeant Draq5 (Thermo Scientific; 62254) was added to the imaging media at 1:1000 ratio. Cells were imaged with a ×40 water immersion objective on the Leica TCS SP8 confocal microscope, which had a live-cell incubation chamber set up to maintain sample at 37 °C and 5% CO₂. Bis-ANS was excited as previously described, and Draq5 was excited using a 633 nm laser and emission was collected from 675 to 725 nm.

MS-5 cells were cultured on 35 mm glass-bottom culture dishes (Greiner Bio-one) for 24 h. JeKo-1 and REC-1 cells with a 10:1 ratio were added to the pre-cultured MS-5 cells and co-cultured for 24 h. Then cells were washed twice with PBS to remove unbound JeKo-1 and REC-1 cells. Then, cells were fixed in 4% (w/v) paraformaldehyde (PFA, Sigma-Aldrich, St. Louise, MO, USA) in PBS for 15 min at room temperature. After washing with PBS, cells were blocked with 5% BSA (Sigma-Aldrich, St. Louise, MO, USA) for 30 min at room temperature. Next, cells were incubated overnight with primary antibodies, mouse anti-CD20 (MS4A1, OriGene, Rockville, MD, USA), and rabbit anti-p65 (PAS-27617, Thermo Fisher Scientific, Bohemia, NY, USA) at 4 °C. After washing with PBS, cells were incubated with secondary antibodies (goat anti-mouse fluorochrome-conjugated Alexa 555, A32727, ThermoFisher, Waltham, MA, USA) and (goat anti-rabbit fluorochrome-conjugated Alexa 488, A32731, ThermoFisher, Waltham, MA, USA) for 1 h at room temperature. After washing with PBS, the cells were mounted using fluoroshield DAPI (F6057, Sigma-Aldrich). Cell imaging was performed using a Nikon A1 confocal microscope with a 20× objective lens and images were analyzed using NIS-elements software (v.5.30.00). For 3D images, Z-stacks with a thickness of 0.97 μm composed of 15–18 images were collected.