Htb 126

Human breast adenocarcinoma MCF7 (ATCC #HTB-22), colon adenocarcinoma COLO 205 (ATCC #CCL-222), lung adenocarcinoma A549 (ATCC #CCL-185) and breast carcinoma Hs 578T (ATCC #HTB-126) cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). COLO 205 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% PenStrep (PS), all purchased from Invitrogen (Grand Island, NY, USA). MCF7 cells were cultured in Eagle's minimum essential medium supplemented with 0.01 mg/mL bovine insulin, 10% FBS, and 1% PenStrep, all purchased from Invitrogen. A549 cells were grown in F-12K medium supplemented with 10% FBS, and 1% PenStrep, all purchased from Invitrogen. MCF7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 0.01 mg/mL bovine insulin, 10% FBS, and 1% PenStrep, all purchased from Invitrogen. Cell lines were incubated at 37°C and 5% CO₂ under humidified conditions, and did not exceed 90% confluence. For capture assays, tumor cells were removed from culture via treatment with trypsin-EDTA (Invitrogen) for 10 min prior to handling. All cells were washed in HBSS, and resuspended at a concentration of 1.0 × 10⁶ cells/mL in HBSS flow buffer supplemented with 0.5% HSA, 2 mM Ca²⁺, and 10 mM HEPES (Invitrogen), buffered to pH 7.4.