Foxp3 fixation

The innate and adaptive immune responses were analyzed at days 0, 1, 3, 5, and 7. Fresh whole blood (150μl) from each study animal was stained with a panel of fluorophore conjugated monoclonal antibodies against the following surface antigens: CD3, CD4, CD8, CD11c, CD14, CD16, CD20, CD28, CD66, CD95, CD123, HLA-DR, and PD1 for 30 minutes at room temperature. Cells were subsequently lysed with 1000μL of FACS lyse (BD Biosciences, USA) then washed twice with 1X FACS buffer. Cells were fixed and the permeabilized with 100μL of FoxP3 fixation (BD Biosciences, USA) for 10 minutes in the dark at room temperature, followed by washing with 1X FoxP3 wash buffer. Intracellular staining was performed with Ki-67 for 45 minutes at 4°C. Fluorescence was measured on the same day using a BD FACSymphony with FACS Diva version 8.0.1 software. Compensation, gating, and analysis were performed using FlowJo (Versions 9 and 10). Antibody/reagent details are given in S5 Table.

The Innate and adaptive immune responses were analyzed at days 0, 1, 3, 5, and 7. Fresh whole blood (150μl) from each study animal was stained with a panel of fluorophore conjugated monoclonal antibodies against the following surface antigens: CD3, CD4, CD8, CD11c, CD14, CD16, CD20, CD28, CD66, CD95, CD123, HLA-DR, and PD1 for 30 minutes at room temperature. Cells were subsequently lysed with 1000μL of FACS lyse (BD Biosciences, USA) then washed twice with 1X FACS buffer. Cells were fixed and the permeabilized with 100μL of FoxP3 fixation (BD Biosciences, USA) for 10 minutes in the dark at room temperature, followed by washing with 1X FoxP3 wash buffer. Intracellular staining was performed with Ki-67 for 45 minutes at 4°C. Fluorescence was measured on the same day using a BD FACSymphony with FACS Diva version 8.0.1 software. Compensation, gating, and analysis were performed using FlowJo (Versions 9 and 10). Antibody/reagent details are given in table S5.

PBMCs (1x10⁶ cells) from each study animal were stained with a panel of fluorophore conjugated monoclonal antibodies against the following surface antigens: CD3, CXCR3, CD20, PD1, CCR6, CXCR5, CCR4, CD4, CCR5, CD69, CD95, CD8, and dump channel (CD20, and live/dead) markers for 30 minutes at 4°C. Cells were subsequently lysed with 1000μL of FACS lyse (BD Biosciences, USA), washed twice with 1X FACS buffer. Cells were fixed and permeabilized with 100μL of FoxP3 fixation (BD Biosciences, USA) for 10 minutes at dark at room temperature, followed by washing with 1X FoxP3 wash buffer. Intracellular staining was performed with Ki-67 and Granzyme B for 45°C minutes at room temperature. Stained cells were washed and re-suspended in FACS buffer. Fluorescence was measured on the same day using a BD FACSymphony with FACS Diva version 8.0.1 software. Compensation, gating, and analysis were performed using FlowJo (Versions 9 and 10). Antibody/reagent details are given in S5 Table.