Cytokine-Producing Cell Profiling by Flow Cytometry

Stimulated blood samples was further stained to determine the cytokine-producing cells by flow cytometry. Generally, after stimulation, whole blood samples were stained with PerCP-Cy5.5 conjugated anti-human CD4 monoclonal antibodies (Cat No. 85-45-0048-42) at room temperature for 20 min, and then incubated with an equal volume of Fix-perm reagent A for 15 min. After washing, Fix-perm reagent B was added. The sample was subsequently incubated with FITC-conjugated anti-IL-17A monoclonal antibodies (Cat No.85-11-7179-42), PE-conjugated anti-IFN- monoclonal antibodies (Cat No. 85-11-7319-82), and eFluro680-conjugated anti-IL22 monoclonal antibodies (Cat No. 85-50-7229-42) for 20 min at room temperature. Cat No.Cat No.Cat No.Antibodies above were all purchased from eBioscience, CA, USA. Fix-perm reagents were purchased from Invitrogen (CA, USA). Samples were washed and immediately detected by BD AccuriC6 Flow Cytometer. Isotopes were applied as negative controls and. Data were analyzed using FlowJo 7.6. Gating strategy could be referred to as previously reported (9 (link)).