Versalyse reagent

Mouse splenic tissue was dissected under deep isoflurane anesthesia and homogenized with 5 mL chilled PBS. The suspension was passed through a cell strainer (100 µm; Falcon). The filtered cell suspension was centrifuged, and the cell pellet was washed twice in PBS. The final pellet was resuspended in 100 µL PBS and added to a DuraClone IM Phenotyping BASIC tube (Beckman Coulter). Mouse splenic lymphocytes were treated with PECy5 anti‐human CD45 antibody (HI30; BioLegend), PECy7 anti‐human CD3 antibody (SK7; BioSource), and FITC anti‐human CD19 antibody (J3‐119; Beckman Coulter) for 20 min on ice followed by perm/wash buffer (BioSource) for permeabilization. Cells were stained with PE anti‐human STING antibody (T3‐680; BioSource). After staining in accordance with the manufacturer's instructions, the cells were exposed to VersaLyse reagent (Beckman Coulter) for 15 min for red blood cell hemolysis, before being fixed with 4% paraformaldehyde in PBS. Lymphocyte phenotyping was performed using flow cytometry with the Gallios instrument (Beckman Coulter).

Freshly EDTA peripheral blood from controls and patients were stained with suitable conjugated antibody: APC-CD8, FITC-CD4, PE-CD28, Pe-Cy-5-CD27, Pe-Cy-5-CD25, PE-CD62L, PE-CD69, Pe-Cy-5-CD14 and PE-CD16 (all Beckmann Coulter, Milan, Italy). Cells were incubated for 15 min at room temperature and for other 10 minute with VersaLyse reagent (Beckmann Coulter) to lyse red blood cells. Data were acquired using a FC500 (Beckmann Coulter) flow cytometer and analyzed using Kaluza software. The area of positivity was determined using an isotype-matched mAb, a total of 10⁴ events for each sample were acquired.