Foxp3 intracellular fixation and permeabilization kit

Cells stimulated in the presence or absence of anti-TLR2 antibody for 24 h were collected after an additional 15 min incubation with 2mM EDTA. The cells were then washed with FACS buffer (PBS with 0.3% BSA and 2mM EDTA) and Fc receptors were blocked with 20 μL of blocking buffer Fc Receptor Binding Inhibitor (eBiosciences) for 10 min at 4 °C. The cells were incubated with 50 μL of antibody cocktail (Supplemental Table 5) for 30 min at RT, washed with PBS and incubated with Fixable Viability Dye eFluor™ 450 (eBiosciences) for 30 min at 4 °C. For intracellular staining, the cells were permeabilized using the FoxP3 intracellular fixation and permeabilization kit (eBiosciences) according to the manufacturer's instructions. Subsequently, 50 μl of intracellular Ab cocktail (Supplemental Table 5) was added and incubated for 30 min at 4 °C. Finally, the cells were washed, resuspended in PBS and kept at 4°C until acquisition on the next day. The cells were acquired on a 12-color LSRII flow cytometer using FACSDiva software (Becton Dickinson, Franklin Lakes, NJ); data analysis was performed using FlowJo™ v10.6.1. Gating strategies are represented in Supplemental Figure 3.