Thymocyte Motility Imaging by Two-Photon Microscopy

CD4SP thymocytes were enriched by magnetic depletion using antibodies against CD8, Gr-1, Ter119, B220, CD25, and CD11b (BioXCell). 1×10⁶Ebi2^+/+ and Ebi2^-/- CD4SP cells were stained with Indo-1AM (Sigma) and CMTPX red (Life Technologies), respectively, for 30min at 37°C,according to manufacturers’ instructions and mixed at a 1:1 ratio in complete RPMI. 3-4 week old pCX-EGFP thymi were embedded in low-melt agarose (Lonza) and vibratome sectioned, with a VT1000S Vibratome (Leica), as previously described [12 (link)]. After incubating thymocytes on slices for 1-2 h at 37°C 5% CO₂, two-photon images were acquired every 15 s, through a depth of 40 μm at 5-μm intervals using an Ultima IV microscope equipped with a 20x NA 1.0 water-immersion objective, and PraireView software (Prairie). MaiTai lasers (SpectraPhysics) tuned to 740 nm and 900 nm were used to excite Indo-1 and EGFP/CMTPX, and emitted light was passed through 400/50, 480/40, 535/50, and 607/45 filters (Chroma Technology) for detection of the two Indo1 emission peaks, EGFP, and CMTPX, respectively. were carried out with Imaris v.8 (Bitplane).