Pe conjugated anti cd4 antibody

The monoclonal antibodies used were allophycocyanin (APC)-conjugated human anti-CD4 (Miltenyi Biotec, Leiden, The Netherlands) and phycoerythrin (PE)-conjugated anti-CD4 antibody (Becton Dickinson, Franklin Lakes, NJ). The stained cells were analyzed for CD4 expression on a MACSQuant flow cytometer (Miltenyi Biotec) or FACS Calibur flow cytometer (Becton Dickinson). GFP⁺ HeLa CD4++ cl1022 cells were sorted with FACS Aria cell sorter (Becton Dickinson) after staining with PE-conjugated anti-CD4 antibody. The purity of the isolated cell fractions was analyzed with FACS Calibur (Becton Dickinson). Analysis of flow cytometry data was performed with MACSQuantify^TM version 2.5 and Flowing software version 2.5.1.

Tumor was digested with 0.25% Trypsin (Sigma-Aldrich) for 45 minutes, followed by flow cytometry analysis and cell sorting. The cell preparations were labeled with PE-conjugated anti-CD4 antibody, APC-conjugated anti-CD25 antibody and Pacific blue-conjugated anti-CD4 antibody (Becton-Dickinson Biosciences, San Jose, CA, USA), GFP was detected by direct fluorescence. Flow cytometry was performed using a FACSAria (Becton-Dickinson Biosciences) flow cytometer, and the data were analyzed and presented using Flowjo software version 10 (Flowjo LLC, Ashland, OR, USA).