Recombinant mouse icam 1 fc

To generate ICAM-1-Fc-F(ab’)₂ complexes (scICAM-1) APC- or PE- labelled anti-human Fcγ specific IgG F(ab’)₂ fragments (Jackson ImmunoResearch) and recombinant mouse ICAM-1 Fc (R&D Systems) were incubated at 4°C for 30 min in PBS as described previously^{30 (link)}.
Primary CD4⁺ T cells were incubated for 30 min at 4°C with 1 μg/ml of anti-CD3ε (2C11). The cells were then activated by crosslinking with anti-Armenian Hamster IgG at 37°C in presence of scICAM-1. For the control experiments the cells were activated with PMA (50 ng/ml) or Mg²⁺/EGTA (10 mM / 1 mM) or CCL19 (250 ng/ml). After the indicated time the reaction was stopped by fixation with 4% PFA for 5 min. The samples were then washed in PBS+10% FCS, stained with PE- or APC-labelled anti-CD4 and analysed on a FACS-Calibur.
For LFA-1 clustering experiments, T cells were incubated with anti-CD3ε for 30 min on ice. Cells were then washed and cross-linked with anti-Armenian Hamster IgG for 30 min at 37°C. Cells were fixed with 4% PFA, washed and block with 10% FCS-PBS for 30 min. Cells were then stained for anti-CD11a and imaged on a Olympus FV1000 microscope fitted with a Olympus Plan super Apochromat 60×/1.4 NA oil objective.