Cd45ra fluorescein isothiocyanate fitc

Intracellular staining for the cytokines Interferon-γ (IFN-γ), Tumor necrosis factor α (TNF-α) and IL-2 was performed as originally mentioned by Jung et al. [59 (link)]. Briefly, cells after one week MLPC were restimulated with appropriate antigens in the presence of Brefeldin A for six hours (Biolegend, San Diego, CA, USA). Cells were harvested for live-dead NEAR (Invitrogen, Thermo Fisher Scientific, Waltham, MA USA) and surface marker staining followed by fixation using paraformaldehyde (PFA, Sigma Aldrich). For surface marker staining the following antibodies were used: CD3 Alexa fluor700 (Biolegend, Fell, Germany), CD8 Peridinin chlorophyll (PerCP, Biolegend), CD197 (CCR7) phycoerythrin Cy7 (eBioscience, San Diego, CA, USA) and CD45RA fluorescein isothiocyanate (FITC, BD Biosciences, Heidelberg, Germany). After permeabilization cells were blocked by FcR blocking reagent (Miltenyi Biotec) then stained for intracellular cytokines using anti-IFN-γ PE, anti-TNF-α Pacific Blue and anti-IL-2 AmCyan antibodies (BD Biosciences, Heidelberg, Germany). Samples were measured with the flow cytometer BD LSR II and analyzed using the BD FACSDiva analysis software (BD Biosciences).

Freshly obtained human lymphocytes were stained with the following fluorescent antibodies against human leukocyte surface markers: CD4-peridinin-chlorophyll-cyanine 5 (PerCP-Cy5.5), CD25-phycoerythrin (PE) or -allophycocyanin (APC), CD45RA-fluorescein isothiocyanate (FITC), CCR7-PE, CCR5-FITC, CCR4-PE-Cy7, CD62L-PE, Ki67-PE or -APC from (BD Biosciences, USA), and LAP (PE and APC) from (R&D Systems, USA). Intracellular staining with Foxp3-APC or -FITC and CTLA-4-APC antibodies was performed after treatment with fixation and permeabilization buffers (eBiosciences, USA), according to the manufacturer's protocol. Intracellular staining for granzyme B, perforin, and TGF-β was performed after stimulation with PMA and ionomycin for 5 h with Golgi stop. The stimulated cells were first reacted with antibodies against the surface markers CD4 and LAP, then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences, USA), and finally stained with TGF-ß-PE, granzyme B-FITC, or perforin-PE antibodies. The fluorescence intensity was analyzed using BD FACSCalibur (BD Biosciences, USA) and FlowJo software (Tree Star, USA).