Cd4 qdot 605

PBMC at 1 × 10⁶ cells in 100 µl R10 were stimulated with peptide pools (F+G+H = NS3/4, I+L+M = NS5A/B) or PMA (phorbol 12-myristate 13-acetate)/ionomycin (50 and 500 ng/ml respectively), or unstimulated (DMSO). Brefeldin-A was added (10 µg/ml) 1 h later, cells were incubated overnight (37 °C), stained with fixable-NIR live/dead dye (Life Technologies), fixed (1% paraformaldehyde) and permeabilised (Foxp3 Fix/Perm kit, BD Biosciences) then stained with the following antibodies at room temperature for 30 mins: CD3-PO (Pacific Orange; Invitrogen, UCHT1), CD4-Qdot 605 (Invitrogen, S3.5) CD8-PB (Pacific Blue; BD biosciences, RPA-T8), IFNγ-Alexa Fluor 700 (BD biosciences, XMG1.2), IL-2-APC (BD biosciences, 5344.111), and TNFα-PE-Cy7 (BD biosciences, MAb11). Flow cytometry was performed using a BD LSRII and analysis by FlowJo (Tree Star) version 10.4.1. All ICS data are corrected for background (cytokine production in paired DMSO wells).
Pestle version 1.8 and Spice version 6.0 were used for background subtraction, data formatting, and data visualization for polyfunctionality assessment of ICS data. Samples with a total T cell response to HCV NS of <0.025% of CD4⁺ or CD8⁺ T cells were excluded. Pie base, means.

Antibodies used were as follows: CD56 BV421, CD3 PE-Cy7 or APC-Cy7, CD14 APC-Cy7, CD19 APC-Cy7, CD57 Pacific Blue or FITC, CD62L PE-Cy7, CD107a PE-Cy7, CD244 PE-Cy5.5, Perforin Pacific blue, CD160 Alexa Fluor 647 (Biolegend), CD16 eFluor450 or FITC, CD69 FITC, CD94 FITC, CD8a PerCP-Cy5.5 (eBioscience), CD161 PE or APC, CD56 APC, IFNγ FITC, NKp30 APC, NKp46 APC, NKp80 APC (Miltenyi Biotec), CD3 Pacific Orange, CD4 Qdot 605, Granzyme B APC (Invitrogen), NKG2C Alexa Fluor 488 or PE, NKG2D PE, PLZF APC, Granzyme A FITC (R&D Systems), CD56 FITC or PE-Cy7, CD85j FITC, IFNγ Alexa Fluor 700, Ki67 FITC (BD Biosciences), Granzyme K FITC (Immunotools), NKG2A PE, NKp44 PE, CD158e1/e2 PE (Beckman Coulter), Phosphatidylserine Alexa Fluor 488 (Merck Milipore). The viability dye Live/Dead fixable Near-IR (Invitrogen) was used in all experiments. Anti-KLRG1 FITC was kindly provided by H. Pircher. Data were acquired on LSRII (BD Biosciences) and analyzed using FlowJo (Treestar, Inc.).

To examine the cellular infiltrate in the ear 48 h after challenge, flow cytometric analysis was performed on infiltrating cells in the ear. Briefly, the inflamed ear was divided into dorsal- and ventral halves. Using a scalpel, the dermis was separated from epidermis and both parts were subsequently incubated with 2000 U/mL collagenase (Sigma) and 2000 U/mL DNAse (Roche, Basel, Switzerland) for 60 min. Next, ear tissue was passed through a 70 μm cell strainer before cells were washed and re-suspended in PBS (w/o Mg²⁺ and Ca²⁺, Gibco/Invitrogen). The cells were counted and cell suspensions were thereafter blocked with anti-CD32/CD16 (Fc block, BDBiosciences) for 10 min and stained with the following anti-mouse mAbs: CD8 APC (BDBiosciences), CD4 Qdot605 (Invitrogen), CD45 efluor450 (eBioscience), TCRβ PECy7 (Biolegend), CD19 PerCPCy5.5 (BDBiosciences), CD88 PE (Biolegend), Ly6G/Ly6C FITC (BDBiosciences), CD11b AF700 (eBiosciences) for 30 min. Flow cytometric analysis of samples was analyzed as described above.