Pe conjugated mouse anti human ifn γ

Brefeldin A (5 μg/mL) was added into the in vitro culture of PBMC for 4 hours. The cell was fixed by 2% paraformaldehyde and permeabilized by saponin (0.5%). Intracellular cytokine was analyzed by cytofluorography employing a Beckman Coulter NAVIOS flow cytometer (Beckman Coulter Co.) after the intracellular cytokine was stained by PE‐conjugated mouse anti‐human IFN‐γ (1/50×; PharMingen).

Cell surface and intracellular staining were conducted as previously described (28 (link)). The experiments used the following reagents: CFDA-SE, PE-conjugated mouse anti-human IFN-γ (BD Pharmingen), APC-conjugated rat anti-human IL-2 (BD Pharmingen), PE-Cy™7-conjugated mouse anti-human TNF-α (BD Pharmingen), APC-conjugated rat anti-human IL-10 (BD Pharmingen), FITC-conjugated mouse anti-human CD4 (BD Pharmingen), Alexa Fluor^® 647-conjugated mouse anti-human Foxp3 (BD Pharmingen), APC-conjugated mouse anti-human CD4 (BioLegend), APC-conjugated mouse anti-human CD8 (BioLegend), PE-conjugated mouse anti-human PD-1 (BD Pharmingen), FITC-conjugated Annexin V (BD Pharmingen), and an Annexin V PE Apoptosis Detection Kit I (BD Pharmingen) with the matched isotype controls. Fluorescence was evaluated via FACS analysis using a BD Accuri™ C6 system within one hour of cell staining. The data were analyzed using FlowJo 7.6 software (Tree Star).