Anti cx3cr1 pe

All cells were assessed for viability using a fixable viability dye (ebioscience eFluor506). Flow cytometric analysis of human and murine cells was performed using >50 antibodies and appropriate directly conjugated mouse, rat and hamster isotype controls. The clones . relevant to the majority of work are as follows: mouse anti-CD3-FITC (Miltenyi; REA641) anti-CD8a-PerCP (Miltenyi; 53-6.7) anti-CD28-PE (Miltenyi; 37.51) anti-KLRG1-APC (Miltenyi; 2F1). Human anti-CD3 APC-Cy7 (biolegend; HIT3a) anti-CD8-PE-Vio770 (Miltenyi; REA734) anti-CD28-eFluor450 (ebioscience; CD28.2) anti-KLRG1-AF488 (a generous gift from Professor Hanspeter Pircher, University of Freiburg) anti-CD57-Vioblue (Miltenyi; TB03) anti-CD244-FITC (biolegend; C1.7) anti-CX3CR1-PE (ebioscience; 2A9-1), and anti-IL-10-FITC (ebioscience; BT-10). For intracellular cytokine staining, cells were cultured with PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin (3 µM; all from Sigma) for 4 h at 37C. Cells were stained for cell surface markers, then fixed and permeabilized in Cytofix/Cytoperm (BD) before intracellular detection of cytokines. Cells were acquired using a CyAn ADP (Beckman Coulter) flow cytometer and analysed using Summit software (software version 4.3; Beckman Coulter). Percentages and mean fluorescence intensity (MFI) were calculated against appropriate isotype controls.