Cd4 fitc

Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of colon cancer patients by density-gradient centrifugation with Ficoll. Then, CD4⁺ T cells were separated from PBMC using Dynabeads™ CD4 Positive Isolation Kit (Thermo Fisher Scientific) according to the manufacturers’ instruction. The purity of the sorted CD4⁺ T was verified by flow cytometry. CD4⁺ T cell suspension was stained with CD4-FITC (Abcam) at 4 °C for 30 min. Then, CD4⁺ T cells were gated with CD4 and SSC, the purity of CD4⁺ T cells was examined using a FACSCalibur (BD Biosciences).
CD4⁺ T cells and Tregs were cultured in RPMI 1640 medium containing 10 % FBS and 1 % penicillin/streptomycin at 37 °C and 5 % CO₂. For activation of CD4⁺ T cells, sorted naive CD4⁺ T cells were activated with anti-CD3 and anti-CD28 (BD Biosciences). For differentiation of Tregs, CD4⁺ T cells were incubated with TGF-β (5.0 ng/mL) for 5 days. Tregs were stained with CD25-FITC and Foxp3-PE antibodies to estimate the Treg differentiation efficiency by flow cytometry. Subsequently, Tregs were transfected with LV-IRF4, LV-ctrl, LV-sh-IRF4 or LV-shRNA. Then, the cell culture medium of these modified Tregs was collected by centrifugation, and then incubated with SW480 or HCT116 cells.

Whole blood cells or PBMC were labeled using the following monoclonal antibodies: CD3-PC5 (UCHT1, Beckman Coulter-BC, Paris, France), CD4-FITC (13B8.2, BC), CD8-FITC (B9.11, BC), CD28-PE (B-T3, Abcam, Cambridge, UK), CD25-PE (M-A251, Becton Dickinson-BD Biosciences-Pharmigen, San Diego, CA, USA), CD56-PE (N901, BC), HLA-G-PE (MEM-G/9, Abcam), and PE-Cy5 Conjugated anti-human Foxp3 (PCH101, eBiosciences, San Diego, CA, USA). All procedures were performed according to the manufacturers’ instructions. For intracellular staining of Foxp3, the cells were labeled with CD4-FITC and CD25-PE antibodies and were stained intracellularly for Foxp3, after fixation and permeabilization. Whole blood samples were treated with BD Pharm Lyse buffer 1× (BD) for 15 min, after staining, to lyse red blood cells.
Flow cytometry was performed on an EPICS Coulter XL-MCL Flow Cytometer (BC). At least 20,000 events were acquired for extracellular and 100,000 events for intracellular staining. Data analysis was performed using the FlowJo V7.5 software (Tree Star Inc., Ashland, OR, USA).