Cgas antibody

Cells were seeded on the tissue culture-treated coverslips (Solarbio) in a 24-well plate. When reached approximately 50% confluency, cells were washed with cold PBS and fixed with cold methanol at -20°C for 10 minutes. After being washed three times with PBS, the cells were blocked with 3% bovine serum albumin in PBS for 1 hour. The coverslips were then probed with Pico488 dsDNA quantification reagent for 1 hour and stained with γH2AX antibody (phospho S139, ab26350, Abcam) and cGAS antibody (#79978, Cell Signaling Technology) overnight. After being washed three times with PBS, the cells were incubated with the secondary antibody Alexa Fluor 555 (ThermoFisher) or Alexa Fluor 488 (ThermoFisher) for 1 hour. Coverslip was mounted with mounting medium containing DAPI (ZSGB-BIO), and the images were captured by confocal microscopy (Perkin Elmer).

For detection of micronuclei by microscopy, 20,000 cells were seeded in an 8-well Ibidi chamber (Ibidi, Gräfelfing, Germany) and incubated for 24 h. Cells were fixed with 4% PFA, washed and stained with DAPI. Images (40x magnification) were taken using a confocal microscope (Zeiss, LSM-880) by a person blinded as to the conditions. For the detection of extranuclear chromosomal fragments by flow cytometry, 20,000 cells in 200 µl of medium were seeded in a 48-well plate and incubated at 37 °C for 72 h. The staining was performed according to the protocol In Vitro Microflow (Litron Laboratories, New York). A FACSCanto II flow cytometer was used to acquire the results using FACSDiva Software and the template In Vitro Microflow by Litron Laboratories for FACSDiva v.6.1.1. FlowJo VX was used for analysis and the gating was performed according to the manufacturer’s protocol. For chromosome missegregation experiments, 30,000 cells were seeded in an Ibidi chamber and incubated for 24 h. Cells were fixed with 4% PFA, washed and stained with anti-alpha-tubulin (Sigma, #T9026) and DAPI. Images were taken under blinded conditions using a confocal microscope (Zeiss, LSM-880). For the detection of cGAS at micronuclei, fixed cells were permeabilized with 0.2% Triton-X, followed by incubation with a cGAS antibody (Cell Signaling, #79978).