Anti cd45 ra fitc

Six days after co-culture, CD8⁺ T cells were harvested and stained to assess CD107a expression following stimulation with PMA (25 ng/ml, Sigma-Aldrich) and ionomycin (1 µM, Sigma-Aldrich) for 3 hr. Cells were stained with anti-CD8-PerCP-Vio700, anti-CD45-RA-FITC, or isotype controls (Miltenyi Biotech, Paris, France). For membrane expression analysis, cells were then stained with anti-CD107a-PE or isotype control (BD Biosciences, San Jose, CA). For total expression analysis, cells were fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter), and stained with anti-CD107a-PE or isotype control. FACS data were acquired using a Canto II 4-Blue 2-Violet 2-Red laser configuration (BD Biosciences).

PB mononuclear cells were isolated by Pancoll density centrifugation (PAN-Biotech, Aidenbach, Germany). Leukemic cells were isolated as CD3⁺CD8⁺CD57⁺ T cells by fluorescence-activated cell sorting (FACS, ARIA III, BD Biosciences; Heidelberg, Germany). T cells from healthy donors were initially enriched (> 98% purity) for either CD4⁺ or CD8⁺ T cells by magnetic cell separation using the MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Afterward, the T cell subsets from healthy donors were isolated by FACS using the following strategies: CD8 naïve T cells: CD8⁺CD45RA⁺CD27⁺, CD8 central memory T cells: CD8⁺CD45RA⁻CD27⁺, CD8 effector memory T cells: CD8⁺CD45RA⁻CD27⁻, CD4 naïve T cells: CD4⁺CD45RA⁺CD27⁺, CD4 central memory T cells: CD4⁺CD45RA⁻CD27⁺, and CD4 effector memory T cells: CD4⁺CD45RA⁻CD27⁻. FACS confirmed a 90–99% purity of the T cell subsets. The following antibodies were used: anti-CD3-PerCP, anti-CD4-PE, anti-CD8-APC-Cy7, anti-CD27-APC, and anti-CD45RA-FITC (all Miltenyi Biotech). Genomic DNA was extracted from all samples using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany).
Analysis of differential gene expression by reverse transcription real-time PCR (qPCR) and Pyrosequencing is described in Additional file 11.