Cell Aggregation Assay

Stably-transfected or wild type 293TA or 293NC cells were labeled with Green Cell Tracker (ThermoFisher/Invitrogen), and trypsinized in 0.05% (w/v) trypsin (ThermoFisher/Invitrogen)/Hanks’ balanced salt solution supplemented with 20 mM HEPES, pH 7.4 (HBSS) in the presence of 1 mM EDTA (calcium independent assay) or 1 mM CaCl₂ (calcium-dependent assay) for 30 min at 37°C. The reaction was stopped by adding the same volume of 0.1 mg/ml soybean trypsin inhibitor (T6522, Sigma, St. Louis, MO, USA) and 10 μg/ml deoxyribonuclease I (DN25, Sigma) in ice-cold DMEM, completely dissociated, and harvested at 1200× g for 5 min at 4 degrees. Aggregation assays were carried out in 24-well non-tissue culture plastic plates that had been precoated with 0.5% BSA/HBSS for at least 2–3 h. In each well, 10⁶ dissociated cells were mixed in 1 ml of HBSS containing 0.5% (w/v) BSA, 1 μg/ml deoxyribonuclease I, and rotated at room temperature. The reaction was stopped by adding 1 ml of 4% (w/v) paraformaldehyde (PBS), and wells were imaged. The ratio of total aggregates and total cells was measured in 10 areas per well.