Alexa fluor 647 antirabbit antibody

Purified CD4⁺ T-lymphocytes from healthy individuals were separated from PBMCs using the CD4⁺ T-cell Isolation Kit II (Miltenyi Biotech) and stained with either 0.25 μmol CFSE or 0.5 μmol Cell Trace Violet (Invitrogen). Purified CD4⁺ T cells (1 × 10⁶) were cultured in RPMI-1640 (Life Technologies) containing l-glutamine, 10% fetal calf serum and antibiotics (penicillin and streptomycin) on a precoated 48-well plate with anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) (Beckman Coulter, Villepinte, France). After 72 h, cells were stained with Vioblue anti-CD3, APC-Vio770 anti-CD4 (Miltenyi Biotech) together with the Aqua Live/Death Vivid detection kit (Invitrogen). Samples were fixed and permeabilized using the FoxP3 permeabilization kit (eBioscience) and further stained with either Alexa Fluor 488 anti-SAMHD1 (a gift from O. Schwartz) or rabbit anti-SAMHD1 (Euromedex) followed by Alexa-Fluor 647 antirabbit antibody (Invitrogen). Fluorescence intensities were measured as described previously. Alternatively, CFSE-labeled cells were sorted according to CFSE fluorescence intensity using a MoFlo Legacy (Beckman Coulter) for mRNA quantification.

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).