Pe cf594 anti cd45ro

Multicolor flow cytometry was performed on fresh PBMCs and frozen LNMCs. T-cell subpopulations were determined using the different combinations of the following fluorochrome-conjugated antibodies: Pacific Blue, PE-Cy7 or Alexa-Fluor 700anti-CD3, PerCP or Alexa-Fluor 700 anti-CD4, PE anti-CD27, PE-Cy7 anti-CCR7, PE-CF594 anti-CD45RO, Alexa-Fluor 488 anti-CXCR5, Alexa-Fluor 647 anti-CCR4, PE anti-CXCR3, PE-Cy7 anti-CCR6 (Becton Dickinson Biosciences, Pont de Claix (Le), France). Vioblue anti-CD3, APC-Vio770 anti-CD4, APC-Vio770 anti-CD45RO, APC anti-CD28 (Milteniy Biotech, Paris, France). Brillant Violet 421 anti-CD279 (Biolegend, London, UK).
Intracellular staining was carried out using the FoxP3 permeabilization solution kit according to the manufacturer's instructions (eBioscience, Paris, France) together with PE Ki67, PE anti-Bcl-6 (BD Pharmingen) or PE anti-FoxP3 (Biolegend), and Alexa-Fluor 488 anti-SAMHD1 (a gift from O. Schwartz) or rabbit anti-SAMHD1 (Euromedex, Souffelweyersheim, France) followed by Alexa-Fluor 647 antirabbit antibody (Invitrogen, Life Technologies, Saint Auben, France).
Dead cells were excluded using the Live/Death Vivid detection kit labeled with an aqua dye (Invitrogen). Fluorescence intensities were measured with an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo version 7.6.5 (Tree Star Inc., Ashland, Oregon, USA).