Antibodies used to detect cell surface markers of PBMCs were: anti-human CD3-Alexa Fluor 700, anti-human CD4-FITC, anti-human CD25-APC-eFluor 780, anti-human CD39-PE-Cy7 and anti-human CD56-PE from eBioscience; anti-human CD8-Pacific Blue, anti-human CXCR3 PE-Cy5 and PE-Cy5 mouse IgG1, κ isotype control from BD Bioscience.
Cells were resuspended in 5 μl Live/Dead solution (Invitrogen-Life Technologies) for 10 min at RT, then incubated with cell surface antibodies for 30 min at RT followed by intracellular staining for Foxp3 using the anti-mouse/rat/human FOXP3 (Biolegend) following the manufacturer's instructions. Flow cytometry analysis was performed using a LSRII (BD Bioscience) and data analyzed with FlowJo software.
Cells were resuspended in 5 μl Live/Dead solution (Invitrogen-Life Technologies) for 10 min at RT, then incubated with cell surface antibodies for 30 min at RT followed by intracellular staining for Foxp3 using the anti-mouse/rat/human FOXP3 (Biolegend) following the manufacturer's instructions. Flow cytometry analysis was performed using a LSRII (BD Bioscience) and data analyzed with FlowJo software.