Alexa fluor 488 nm fluorophore conjugated secondary antibody

The cells grown on glass coverslips were treated with cGAMP for 2 h or left untreated following fixation with 4% PFA for 40 min and 20-min permeabilization with 0.2% Triton X-100. Next, blocking solution (2% goat serum (Sigma) in DPBS) was applied for 1 h, and afterwards samples were incubated with rabbit anti-SAM68 (Cell signaling) (1:100 dilution), mouse anti-GM130 (BD biosciences) (1:100 dilution) and sheep IgG STING/TMEM173 (R&D Systems) (1:50 dilution) antibodies in blocking solution for 1 h at room temperature. After three washes with DPBS, 5 min each, the cells were stained with donkey anti-sheep Alexa Fluor 488 nm fluorophore-conjugated secondary antibody (1:500, Invitrogen), donkey anti-rabbit Alexa Fluor 568 nm fluorophore-conjugated secondary antibody (1:500, Invitrogen), donkey anti-mouse Alexa Fluor 647 nm fluorophore-conjugated secondary antibody (1:500, Invitrogen) and counterstained with PureBlu DAPI Dye (1:100, Bio-Rad) for 1 h at room temperature in the dark. The cells were then washed three times with DPBS and mounted onto microscope slides using ProLong Gold Antifade mountant (Invitrogen). Slides were air-dried in the dark and examined on the next day using a Zeiss LSM 800 Inverted Confocal Microscope with corresponding Zeiss Zen software.

THP1 cells (WT and SAM68 KO) were seeded onto glass coverslips placed on the bottom of a 12-well plate and treated with PMA for further differentiation, as described above. After 24-h incubation, cells were stimulated with cGAMP for 5 h or left untreated for control studies. Afterward, cells were treated for 40 min with 4% PFA following a 20-min permeabilization using 0.2% Triton X-100 in DPBS. Next, blocking with 2% FCS in DPBS was performed for 40 min, and rabbit cleaved caspase 3 antibody (1:400, Cell Signaling) or rabbit IFIT1 antibody (1:800, Cell Signaling) were applied for 1 h at room temperature. After three washes with DPBS, 5 mi each, the cells were incubated with goat anti-rabbit Alexa Fluor 488 nm fluorophore-conjugated secondary antibody (1:400, Invitrogen), Alexa Fluor Plus 647 Phalloidin (1:400, Invitrogen), and PureBlu DAPI Dye (1:100, Bio-Rad) for 1 h at room temperature in the dark. The cells were then washed three times with DPBS and mounted onto microscope slides using ProLong Gold Antifade mountant (Invitrogen). Slides were air-dried in the dark and examined on the next day using a Zeiss LSM 710 Inverted Confocal Microscope with corresponding Zeiss Zen software. Cleaved caspase 3-stained area was measured using ImageJ software.