Magnetic separator

Subtraction enrichment was performed according to the manufacturer’s instructions (Cytelligen, San Diego, CA, USA) similar to previously published methods with minor modifications [13 (link)]. Briefly, 6 ml of peripheral blood was collected into a tube containing acid citrate dextrose (ACD) anti-coagulant (Becton-Dickinson, Franklin Lakes, NJ, USA). Blood samples were centrifuged at 200×g for 15 min at room temperature to remove plasma. Sedimented blood cells were gently mixed with 3 ml of hCRC buffer, loaded on the non-hematopoietic cell separation matrix in a 50 ml tube, and subsequently centrifuged at 450× g for 5 min. The middle layer containing white blood cells (WBCs) and tumor cells was collected into a 50 ml tube and subsequently incubated with 300 µl of anti-CD45 monoclonal antibody-coated magnetic beads at room temperature for 20 min with gentle shaking. WBCs bound to magnetic beads were depleted using a magnetic separator (Promega, Madison, WI). The bead-free supernatants were collected into a 15 ml tube, followed by adding hCTC buffer to 14 ml. Samples were then spun at 500×g for 4 min at room temperature. Supernatants were aspirated down to 100 µl and resuspended, mixed with the cell fixative (Cytelligen), then applied to formatted slides, and dried for subsequent iFISH analyses.

The SiMAG-Carboxyl (0.5 µm ϕ, Chemicell. Prod. No. 1402) particles were washed twice with 1 M MES buffer using a magnetic separator (Promega). After the second wash, the magnetic particles were resuspended in MES buffer containing 10 mg EDC (Thermo Fisher). Subsequently, only freshly prepared EDC was added to the particles, followed by mixing on a shaker for 10 min at RT. After this step, the EDC solution was removed and replaced by prepared NHS (Thermo Fisher), followed by fully mixing on a shaker and reaction at RT for 30 min. Then, the NHS was removed and an amine group containing ligand GABA (10 mg dissolved in double distilled water) was added to the activated particles and mixed on a shaker for 2-3 h at RT. Next, the particles were resuspended in blocking buffer (10 mm hydroxylamine [Thermo Fisher]), shaken, and reacted for 10 min at RT to terminate the reaction. Finally, the particles were resuspended in PBS for storage and later use.