Single-cell suspensions were plated and stained for 20 min at 4 °C with a combination of fluorescently labeled antibodies specific for surface markers including CD8α (53–6.7), CD25 (PC61.5), CD11a (M17/4), CD43glyco (1B11), CD44 (IM7) CD49a (Ha31/8), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), CD122 (5H4), CXCR3 (CXCR3–173), CXCR6 (SA051D1), CX3CR1 (SA011F11), KLRG1 (2F1), Thy1.1 (OX-7 or HIS51) and fixable viability stain (BD Horizon). Antibodies were purchased from BD Bioscience, BioLegend, eBioscience/Thermo Fischer Scientific, or Tonbo biosciences. Cells were fixed with BD Cytofix (BD Biosciences).
To asses cytokine production, single cells suspensions were plated in the absence (no stim) or in the presence 1 μM OVA(257–264) peptide in the presence of GolgiPlug (BD Bioscience) for 5 h at 37 °C or detected directly ex vivo in the absence of GolgiPlug. Cells were stained for surface molecules including fixable viability stain (BD Horizon), fixed and permeabilized using the transcription factor staining buffer kit (Thermo Fisher Scientific) and stained for cytokines including IFN-γ (XMG1.2), TNF (MP6-XT22), and IL-2 (JES6–5H4).
All samples were acquired using LSRFortessa (BD Bioscience) and analyzed using FlowJo software, version 9.9.4 (FlowJo LLC).
To asses cytokine production, single cells suspensions were plated in the absence (no stim) or in the presence 1 μM OVA(257–264) peptide in the presence of GolgiPlug (BD Bioscience) for 5 h at 37 °C or detected directly ex vivo in the absence of GolgiPlug. Cells were stained for surface molecules including fixable viability stain (BD Horizon), fixed and permeabilized using the transcription factor staining buffer kit (Thermo Fisher Scientific) and stained for cytokines including IFN-γ (XMG1.2), TNF (MP6-XT22), and IL-2 (JES6–5H4).
All samples were acquired using LSRFortessa (BD Bioscience) and analyzed using FlowJo software, version 9.9.4 (FlowJo LLC).