Lab tek 2 8 well chambered coverglasses

Lab-Tek II 8–well chambered coverglasses (Nunc) were coated with 3 µg/ml of recombinant human ICAM-2/Fc chimera (R&D Systems) overnight at 4°C and rinsed with PBS before use. Alternatively, collagen-coated slides were stored at 4°C until use (BD). 200 µl of cycling T cells (10⁵/ml) were gently added into each well. After 1 h incubation at 37°C in the presence of humidified 5% CO₂, time-lapse DIC microscopy and analysis was performed as described above in Collagen gel migration assays for morphological analyses.

Human foreskin tissues discarded after routine newborn circumcisions were stored in HBSS at 4°C for 3–7 d before use. Tissues were warmed to 37°C overnight in PBS supplemented with 10% FBS, cut into 5 × 5 mm pieces, and the epidermis carefully peeled off using a small forceps. T cells, labeled with 0.5 µM CellTracker Green CMFDA (5-chloromethylfluorescein diacetate; Life Technologies), were dropped onto the exposed dermis and allowed to migrate into the tissues at 37°C for 3 h. Cells remaining on the dermal surface were gently rinsed away, and tissues immersed in complete medium within Lab-Tek II 8-well chambered coverglasses (Nunc). Live cell imaging was performed at 37°C in the presence of 5% CO₂, using a TCS SP5 laser scanning confocal microscope (Leica) with excitation at 488 nm. Image stacks were collected with a 4-µm vertical step size at a depth of 40–60 µm. Collagen was visualized by refractive imaging.