Celltrackers

SU-8 2050 and its developer were purchased from MicroChem Corp. (USA) and used for microfabrication of microfluidic device master mold on a silicon wafer obtained from Nano-BAZAR (Iran). SYLGARD^® 184 polydimethylsiloxane (PDMS) kit, and Tygon tubing were purchased from Dow Corning (USA). MCF7 and MDA-MD-231 breast cancer cell lines were purchased from Pasteur Institute (Tehran, Iran). Cell culture reagents including Dulbecco’s phosphate buffer saline (DPBS), Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), trypsin-Ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin, and Geltrex^® were purchased from Gibco (Thermofisher Scientific, USA) while CellTrackers were obtained from Invitrogen (Thermofisher Scientific, USA).

Confluent stably-transfected L cells or transiently-transfected 293T cells were trypsinized in the presence of 1 mM EDTA at 37°C as described previously (Yamagata and Sanes, 2008 (link)). In some experiments, cells were labeled with green or red Cell Trackers (Invitrogen). The reaction was stopped by adding the same volume of 0.1 mg/ml soybean trypsin inhibitor (T6522, Sigma, St. Louis, MO) and 10 µg/ml deoxyribonuclease I (DN25, Sigma) in HBSS supplemented with 20 mM HEPES, pH 7.4. All the cell aggregation assays were carried out in 24-well non-tissue culture plasticwares that had been precoated with 0.5% BSA/HBSS. In each well, dissociated cells were mixed with 1 ml of HBSS containing 0.5% (w/v) BSA, 1 µg/ml deoxyribonuclease I, 20 mM HEPES, pH 7.4, and rotated at room temperature for 30–60 min. The reaction was stopped by adding 1 ml of 4% (w/v) paraformaldehyde/PBS, and observed under fluorescent microscopes.