Tcrβ pe cy7

Single cell suspensions from the spleen, lymph node and thymus were prepared and stained after a washing step for surface marker expression with the following fluorochrome conjugated antibodies: anti-CD3-PECy7, anti-CD4-FITC, anti-CD8-APC and anti-B220-PE, (all from Biolegend). For the staining of activation markers, cells were pre-activated for 24 h with stimulating antibodies (aCD3 and aCD28) and then stained with the following antibodies: anti-CD25-APC, anti-CD44-PECy7 and anti-CD69-PE (all from Biolegend). For analyses of thymocytes the following antibodies were used: anti-CD24-FITC, anti-CD5-PerCP Cy5.5 and TCRβ-Pe Cy7 (all from Biolegend).
For the staining of intracellular FoxP3, the cells were fixed and subsequently permeabilized to the staining of surface antigens. The FoxP3 FITC staining buffer set (eBioscience) was used for the detection of Foxp3. Data were acquired on a FACSCalibur (CellQuest, BD Biosciences) and analyzed with FlowLogic software (eBioscience).

After perfusion spleens, lymph nodes, livers and lungs were harvested. Livers and lungs were digested with 2 mg/ml collagenase D (Roche Diagnostics, Mannheim, Germany) and 100 µg/ml DNaseI (Roche Diagnostics) for 60 min at 37°C prior to single-cell suspension. Blood was collected in tubes containing Na heparin (Ratiopharm). After erythrocyte lysis (5 min in 0.15 MNH₄Cl, 0.02 M HEPES, 0.1 mM EDTA for organs or 10 min in BD FACS Lysing Solution (BD Biosciences) for blood) and FcγR blocking (5 μg/ml rat anti-mouse CD16/CD32; clone 93, eBioscience), cells were incubated in PBS, 2% FCS, 2 mM EDTA for 30 min at 4°C with varying combinations of the following antibodies: CD3-PE (17A2), CD4-PE (GK1.5), TCRγδ-FITC (GL3, BD Biosciences), TCRβ-PE-Cy7 (H57–597, Biolegend, San Diego, CA), CD3-APC (145–2C11), CD4-Alexa Fluor 700 (GK1.5), CD8-Alexa Fluor 700 (53–6.7), CD27-FITC (LG.7F9), NKG2D-PE (CX5), TCRγδ-PerCPeFluor 710 (GL3, eBioscience) and Vγ3/5-FITC (536, BD Biosciences). Antibodies Vγ1-FITC (2.11), Vγ4-FITC (49.2) and Vγ7-FITC (F2.67) were a kind gift from P. Pereira. To determine absolute cell numbers Trucount Beads (BD Biosciences) were added.
FACS analysis was performed on LSRII or FACSCalibur machines (Becton Dickinson) running CellQuest software and analyzed with FACSDiva software or FlowJo (Tree Star, Ashland, OR).

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.