SAMHD1 Intracellular Staining in PBMCs

Intracellular SAMHD1 staining was carried out as previously described9 (link). Briefly, PBMCs were stained for 30 min at room temperature using a mixture of anti-CD14-APC, anti-CD69-APC-Cy7, and anti-CD3-PB antibodies. After the cells were washed, they were fixed for 30 min at room temperature using BD Cytofix Fixation Buffer. Cells were permeabilized in BD Phosflow Perm Buffer III for 2 min at 4 °C in the dark and then were washed twice. Cells were then stained for 1 h with rabbit anti-human SAMHD1 polyclonal antibodies (1:100, Proteintech), followed by a mixture of anti-CD4-PE, and donkey anti-rabbit Alexa Fluor 488, antibodies (1:200, Invitrogen), for an additional 1 h. After the washes, cells were detected using a BD FACSAria™ II Flow Cytometer. The antibodies used for flow cytometric analysis were PE-CD4 (Biolegend, PRA-T4), APC-CD14 (Biolegend, HCD14), Pacific Blue-CD3 (Biolegend, UCHT1), APC-Cy7-CD69 (Biolegend, FN50), rabbit anti-human SAMHD1 pAb (Proteintech, 12586-1-AP), and Alexa Fluor^® 488 donkey anti-rabbit IgG (H+L) (Invitrogen, A-21206). Data were analyzed using FlowJo 7.6 software.