Cell preparation and adoptive transfer were performed as previously described23 (link). Briefly, splenic DCs were purified from B10.A CD45.2− mice using CD11c microbeads (Miltenyi Biotec). Polyclonal CD4+ T cells from LNs of B10.A CD45.2− and TCR transgenic 5C.C7 CD4+ T cells from WT or IL2−/− B10.A CD45.2+ 5C.C7 TCR-transgenic RAG2−/− mice were purified by negative immunomagnetic cell sorting (Miltenyi Biotec). DCs were incubated in vitro with PCC peptide (10 μM pPCC, American Peptide Company) and LPS (1.0 μg/ml, Invivogen) for 4 hours at 37°C before s.c. injection at 1 × 106/footpad. CD4+ T cells were transferred by i.v. injection at 2 × 106/recipient 18 hours post-transfer of DCs.
For 2P intravital imaging, DCs were stained with 100 μM CTB (7-amino-4-chloromethylcoumarin, Molecular Probes), polyclonal CD4+ T cells were stained with 1.25 μM CMFDA (5-chloromethylfluorescein diacetate, Molecular Probes), and WT or IL2−/− TCR transgenic 5C.C7 CD4+ T cells were stained with 1.25 μM CMTPX (Molecular Probes). 24 hours after T cell transfer, mice were anesthetized with isoflurane and popliteal LNs were surgically exposed. Imaging was performed on a Zeiss 710 microscope equipped with a Chameleon laser (Coherent) tuned to 800 nm in combination with a 20× water-dipping lens (NA 1.0, Zeiss) using Zen 2010 acquisition software.
For 2P intravital imaging, DCs were stained with 100 μM CTB (7-amino-4-chloromethylcoumarin, Molecular Probes), polyclonal CD4+ T cells were stained with 1.25 μM CMFDA (5-chloromethylfluorescein diacetate, Molecular Probes), and WT or IL2−/− TCR transgenic 5C.C7 CD4+ T cells were stained with 1.25 μM CMTPX (Molecular Probes). 24 hours after T cell transfer, mice were anesthetized with isoflurane and popliteal LNs were surgically exposed. Imaging was performed on a Zeiss 710 microscope equipped with a Chameleon laser (Coherent) tuned to 800 nm in combination with a 20× water-dipping lens (NA 1.0, Zeiss) using Zen 2010 acquisition software.