Foxp3 cytoperm cytofix staining kit

Intracellular FOXP3 and cytokines were assayed by flow cytometry, as described [11 (link)]. Before fix/permeabilization, cells were surface-stained at 4 °C for 30 min. Purified lymphocytes were washed with FACS buffer (5% BSA (Sigma-Aldrich, Burlington, MA, USA) and 1× PBS) and then prepared for surface or intracellular staining, as described [11 (link)]. For surface staining, the manufacturer’s recommended amount of fluorescently conjugated antibody specific to the surface marker to be visualized was added to 1 × 10⁶ cells resuspended in 100 μL of FACS buffer for 30 min at 4 °C, then washed twice with FACS buffer prior to being either analyzed or further processed. In the latter, following twice wash with FACS buffer, cells were immediately fixed in 4% paraformaldehyde (PFA) and permeabilized with 90% methanol, followed by staining. For IL-10 intracellular cytokine staining, the FOXP3 Cytoperm/Cytofix staining kit (BD Pharmingen, San Diego, CA, USA) was used after cells were stimulated for 24 h on 1 μg/mL anti-CD3-coated plates with soluble 1 μg/mL anti-CD28 antibody or anti-CD3/CD28 conjugated Dynabeads (Gibco, Thermo Fisher) and treated in culture for 6 h with BD GolgiStop^TM (BD, Franklin Lakes, NJ, USA). T cell proliferation was measured by staining cells with 5 μM CFSE (Molecular Probes, Eugene, OR, USA) before cell culture and analyzing them using flow cytometry.