Purified Hamster Anti-Mouse TCR β Chain

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	Cell suspensions were stained with antibodies for flow cytometry. For intracellular staining, cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before staining with intracellular antibodies. For intracellular cytokine secretion assays, cells were incubated for 4 hr with PMA and Ionomycin in the presence of Brefeldin A.

Protocol tips
Cell suspensions were stained with antibodies for flow cytometry. For intracellular staining, cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before staining with intracellular antibodies. For intracellular cytokine secretion assays, cells were incubated for 4 hr with PMA and Ionomycin in the presence of Brefeldin A.

Publication protocol

T cells were recovered from skin as described (Mackay et al., 2012). Briefly, skin tissue was incubated for 90 min at 37°C in dispase (2.5mg/ml) followed by the separation of epidermis and dermis. Epidermal sheets were subsequently incubated for 30 min in trypsin/EDTA and remaining skin tissue was chopped into small fragments and incubated for 30 min at 37°C in collagenase type 3 (3 mg/ml). For recovery of T cells from the lung, mice were injected i.v. with 3 μg of Alexa Fluor 700-conjugated antibody to CD3 10 min prior to sacrifice, and mice were then perfused before the collection of lung tissue that was digested for 90 min in collagenase type 3 (3 mg/ml). Cell suspensions were stained with antibodies for flow cytometry. For intracellular staining, cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before staining with intracellular antibodies. For intracellular cytokine secretion assays, cells were incubated for 4 hr with PMA and Ionomycin in the presence of Brefeldin A. The following antibodies were purchased from BD PharMingen: anti-CD45.1 (A20), −CD45.2 (104), −Vα2 (B20.1), −CD8α (53-6.7), −TCR-β (H57-597), −Bcl-2 (3F11), and IL-17A (Tc11-18H10). The following antibodies were purchased from eBioscience: anti-CD45.1 (A20), −CD45.2 (104), −CD8α (53-6.7), −CD3 (500A2), anti-CD103 (2E7), −KLRG1 (2F1), CD122 (TM-b1), T-bet (4B10) Eomes (Dan11mag), and RORγt (AFKJS-9); anti-CXCR3 and -TGF-βRII were purchased from R&D.

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