35 mm glass bottom plate

Samples were plated onto 35-mm glass bottom plates (MatTek) precoated with Concanavalin A (Sigma). After 5 min, cells were imaged on a Deltavision widefield fluorescent microscope (GE Healthcare) using an Olympus 60× objective. Images were acquired, deconvoluted, and projected using SoftWoRx (GE Healthcare). Analysis of DNA in the bud neck utilized a plugin for FIJI. At least 200 cells were counted for every time point.

Primary MEFs at the indicated passage were plated onto 35 mm glass bottom plates (MatTek) and at 60 % confluency were fixed with formaldehyde for 20 minutes at 4°C. SA-β-gal staining was performed in the standard fashion. Cells were incubated overnight with freshly made staining solution and the percentage of cells positive for SA-β-gal was calculated from a total of 200 cells per plate. For γH2AX analysis, cells were incubated in rabbit anti-γH2AX antibody (Cell Signaling) for one hour followed by goat anti-rabbit FITC secondary antibody (Vector Laboratories Inc., Burlingame, CA, USA). Glass bottom coverslips were removed and mounted on slides using Fluoro Gel-II mounting medium containing DAPI (Electron Microscopy Sciences). Experiments for SA-β-gal and γH2AX analysis were repeated in triplicate.

Intracellular calcium concentration ([Ca²⁺]i) was measured in single EA.hy926 cells using ratiometric calcium indicator Fura 2-AM (Life Technologies, Carlsbad, CA). Cells grown on 35 mm glass-bottom plates (MatTek, Ashland, MA) were loaded with 1 μM Fura 2-AM in calcium buffer (140 mM NaCl, 2.8 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose and 10 mM HEPES) for 15 min, washed and incubated in calcium buffer for another 15 min. Fura 2-AM-loaded cells were illuminated in calcium buffer by alternating 340/380-nm light delivered every 300 ms by a DG-4 argon exciter (Sutter Instruments, Novato, CA) under the control of MetaFluor software, and fluorescence images were captured at an emission of 510 nm with a Photometrics Cool SNAP HQ charge coupled device camera (Roper Scientific, Tucson, AZ) based on a Nikon TE2000 fluorescent microscope. Calf thymus histones (50 µg mL⁻¹) without or with 3 min of pre-incubation with RNA aptamer KU7 at various concentrations were added to loaded cells and calcium images were recorded for 10 min for each treatment condition. [Ca²⁺]i was reported as the ratio of Fura 2 fluorescence emission at 340 and 380 nm (F340/F380) normalized to baseline. All procedures were performed at room temperature.

Cells were plated in 35 mm glass bottom plates from MatTek Corporation (Ashland, MA, USA). After treatment, cells were washed with 1× PBS and fixed in 4% paraformaldehyde at room temperature for 15 min. Cells were then washed three times with 1× PBS. Blocking was completed in 3% BSA, 0.1% Triton-X100 in 1× PBS. Anti-interferon alpha (Abcam, Cat# ab196221) and Anti-IL-28A (Abcam, Cat# ab233754) antibodies were used to detect IFN-α and IFN-λ proteins, respectively. AlexaFluor 488 donkey anti-mouse (Invitrogen, Cat# A21206) and AlexaFluor 594 donkey anti-sheep (Jackson ImmunoResearch, cat# 713-585-147) were used as secondary antibodies. Antibodies were diluted in 0.5% BSA + 0.05% Triton X100 in 1× PBS. After each antibody incubation, cells were washed three times with 0.05% Triton X100 in 1× PBS. For DNA counterstaining, a 1 µg/mL solution of Hoechst 33342 (Cat# H1399, Sigma-Aldrich, St. Louis, MO, USA) in 1× PBS was used. Immunofluorescence images were acquired with a 100× oil-immersion lens using the Zeiss confocal microscope (LSM700, Opti-Ups 1000B).