Cd45ra v500 clone hi100

Twenty thousand immDCs, chemotherapy-treated HL-60, KG-1 or primary AML cells pulsed DCs (as described in Section 4.6) were co-cultured for 5 days in complete RPMI with 200,000 autologous CD3⁺ T cells at the ratio of 1:10. The CD3⁺ T cells alone were used as negative control. After 5 days of co-culture, T cells were stained for 15 min in the dark using the following anti-human mAbs: CD4 APCH7 (clone SK3; BD Biosciences), CD25 PeCy7 (clone BC96; Biolegend), CD127 PerCP 5.5 (clone A019D5; Biolegend), CD45RA V500 (clone HI100; Biolegend), PD-1 APC (clone EH12.2H7; Biolegend). Intracellular staining of FOXP3 using Foxp3/Transcription Factor Staining Buffer Set (eBioscience/ThermoFisher) was performed as follows. For each sample, unstained CD3 cells were used as negative fluorescence control. At least 5000 events of Total Tregs in each sample were collected and analyzed using a FACS Canto II Flow Cytometer (BD Biosciences).

Fifteen thousand DCs were prepared as described in 2.7 and cocultured with 150,000 autologous CD3⁺ T cells at a concentration of 1 × 10⁶ T cells/ml. After 5 days of coculture, T cells were harvested, washed with PBS and stained for 15 min at the dark using the following anti-human mAbs: CD3 APC-H7 (clone SK7, BD Biosciences), CD4 Pe-Cy7 (clone SK-3, Thermofisher), CD8 PE (clone SK1, BD Biosciences), CD279/PD-1 APC (clone MIH4, Thermofisher), CD197/CCR7 Alexa Fluor 647 (clone G043H7; Biolegend), and CD45RA V500 (clone HI100; Biolegend). Unstimulated CD3⁺ T cells were used as negative control, and unstained CD3⁺ T cells were used as negative fluorescence control. At least 10,000 events of each sample were collected and analyzed at FACS Canto II Flow Cytometer (BD Biosciences).