Icam 1 fc

ICAM-1 Fc (R&D Systems) was adsorbed to a polystyrene Petri dish at 4 or 40 μg/ml in PBS. After a one-hour incubation at 37°C, plates were rinsed with PBS and non-specific binding sites were blocked with 1% human serum albumin (Sigma-Aldrich) for one hour at room temperature. Freshly isolated naïve CD4⁺ T cells (5.5 x 10⁵) were added to Petri dishes in 1.5 ml of L15 media containing 0.5% FBS and 10mM HEPES and supplemented with 200 ng/ml of CCL19 and CCL21. Dishes were incubated for one hour at 37°C and then fixed with 1% paraformaldehyde. Unbound cells were removed after extensive rinsing with PBS supplemented with 0.2% human serum albumin, 1 mM CaCl₂, and 1 mM MgCl₂. Dishes were imaged at 2.5X magnification and the number of adherent cells was counted. For the transwell migration assay, a 96-well modified Boyden chamber with 5 μm pore size was purchased (Neuro Probe). The top membrane was coated with 0, 4, or 40 μg/ml of ICAM-1 Fc in PBS for one hour at room temperature. CCL19 or CCL21 (0.1, 1, or 10 ng/ml) in migration buffer (RPMI 1640, 0.5% FBS, and 100U/ml penicillin-streptomycin) was plated in the bottom chamber. Freshly isolated naïve CD4⁺ T cells (5 x 10⁴) were added to the top chamber and incubated for 4 hours at 37°C. Subsequently, the membrane was removed and cells in bottom chamber were counted with a hemocytometer.

Black 96-well plates with clear bottom (Costar) were pre-coated with 100 μg/ml protein A (Pierce) in PBS overnight at 4°C. Plates were washed in HBSS and coated with 10 μg/ml ICAM-1-Fc (R&D Biosystems) in HBSS for 1.5 h at 37°C. Subsequently, plates were washed and blocked for 1 h at 37°C using HBSS and 0.5% low-fat BSA (A7511, Sigma). Jurkat cells were stained with Vybrant® DiD cell labeling solution (Thermo Fisher Scientific) according to the manufacturer's protocol and stimulated with 0.5 μg/ml CCL19/CCL21 or 10 mM MgCl₂ and let adhere to immobilized ICAM-1-Fc for 20 min in HBSS and 0.5% low-fat BSA. Non-adherent cells were washed off the plates by 3–4 washing steps and cell-associated fluorescence was measured using a Tecan® Spark 1 M microplate reader. Percentage of adherent cells was calculated in relation to unwashed wells (input).

Purified CD4 T cells were seeded into top chambers over 3 μm or 5 μm transwell filters with 100 ng/mL CCL19 (PeproTech) in the bottom chamber. After 1.5 hrs at 37°C, cells were recovered from the lower chamber and counted by high throughput enabled flow cytometer LSR II (BD). Percentage of migrated cells was determined as a percentage of total input. In some cases, the transwell filters were pre-coated with BSA or 2 μg/mL ICAM-1-Fc (R&D Systems).