Quantification of cytosolic DNA

1. Buffers and reagents preparation

• Permeabilization buffer (0.1%Tween 20, 0.01% Triton-X in Phosphate Buffer Saline): Mix 50mL of 1×Phosphate Buffer Saline (PBS) with 50μL of Tween 20 and 5μL of Triton-X.
• PBST: Mix 100mL PBS and 100μL Tween 20.
• 1% BSA-PBST: Dissolve 1g of bovine serum albumin (BSA) in 100mL of PBST.
• Blocking buffer (1% BSA, 0.1% Tween 20, 2.25% Glycine): Dissolve 1.126g of glycine in 50mL of 1% BSA-PBST.

2. Procedure

• Seed 50,000–100,000 cells/well in 1 mL of growth medium.
• Wash the cells with 1× PBS.
• Fix the cells with fresh 4% of para-formaldehyde (4% PFA; Santa Cruz Biotechnology #sc-281692) for 10min at room temperature.
• Discard the 4% PFA in an appropriate container.
• Wash the cells in 1× PBS.
• Incubate the cells with the permeabilization buffer for 7min at room temperature.
• After three additional washes with 1× PBS (for 5min at room temperature), block nonspecific binding sites by incubating the cells with the blocking buffer for 30min at room temperature.
• Remove the blocking buffer (Do not wash).
• Dilute anti-dsDNA antibody (sc-58749, Santa Cruz Biotechnology) at 1:100 in 1% BSA-PBST.
• Incubate your sample with the diluted anti-dsDNA antibody in humidified chamber overnight at 4°C.
• Wash 3 times by 1× PBS (for 5min at room temperature).
• Dilute the secondary antibody (goat anti-mouse IgG H&L Alexa Fluor® 488 preabsorbed, abcam, # ab150117) at 1:200 in 1% BSA-PBST.
• Wash 3 times by 1 × PBS (for 5 min at room temperature).
• Let it sit for 1–3h at room temperature (Note: protect from the light).
• Keep the slide overnight at 4°C in slide box (Note: upside, protect from the light).
• Observe and acquire pictures with a fluorescence microscope using the RFP/GFP and DAPI channels the next day to ensure that the mounting medium is completely dry.
• Slides can be stored at 4°C.
• Upon adherence (16–24 h after seeding, according to cell type), cells are treated if needed.
• Incubate samples with the diluted secondary antibody for 1 h at room temperature (Note: protect from the light).
• Drop mounting media containing DAPI (Vector laboratories, Vectashield® Hardset™ Anti-fade mounting medium with DAPI, #H-1500) on a slide, and put carefully the cover slip.−
• Let it sit for 1–3h at room temperature (Note: protect from the light).
• Keep the slide overnight at 4°C in slide box (Note: upside, protect from the light).
• Observe and acquire pictures with a fluorescence microscope using the RFP/GFP and DAPI channels the next day to ensure that the mounting medium is completely dry.
• Slides can be stored at 4°C.