**2.3. Antibodies**

Antibodies, which were used for multiparameter flow cytometry, were as follows: anti‐CD45‐ PerCP, anti‐CD44‐APC (allophycocyanin, clone G44‐26, catalog number 559942) and anti‐ CD24‐FITC (fluorescein isothiocyanate, clone ML5, catalog number 555427) were from Becton Dickison Crop., USA. Ep‐CAM (epithelial‐cell adhesion molecule) and CK‐8, CK‐18, CK‐19 (CK‐8, CK‐18, CK‐19 phycoerythrin staining) were from Abcam, USA.

### **2.4. Preparation of samples**

Every patient with breast cancer drew 20 ml blood for CTCs detection and three healthy volunteers. The blood of healthy volunteers was treated as negative control. About 5 ml blood was discarded to avoid contamination with skin cells as previously described [26]. We separated mononucleocytes from 15 ml blood by Ficoll‐Paque (Haoyang Biological Production Limited Company, Tianjin, China) for 20 min with 1800 × *g* at 25°C. One half of the mononu‐ clear cells was resuspended in phosphate‐buffered saline (PBS) for multiparameter flow cytometry on the account of at least 2–3 × 106 cells for each sample and the other half was kept in Trizol reagent (Invitrogen, UK) at -70°C until RNA extraction for RT‐PCR.

#### **2.5. Flow cytometry**

Mononucleocytes were enriched and washed twice with PBS and then labelled antibodies that target white cell antigens and epithelial cell antigens (CD45‐, Ep‐CAM<sup>+</sup> , CK‐8, CK‐18, CK‐ 19<sup>+</sup> ), kept in dark at 4°C for 30 min. We added 20 μl monoclonal antibodies for each sample. Cell pellets were resuspended in 250 μl PBS and enumerated by FACS Caliber™ (Becton Dickison Crop., USA) at last.

SKBR‐3 breast cancer cells were used to evaluate the sensitivity of the flow cytometry (tumor cells recovery). Tumor cells were resuspended and counted. 1, 10, 50, 500 cells were spiked, respectively, into 7.5 ml of blood from healthy person. And then, it was processed as described as earlier and control the preparation for the same volume of the cell suspension and cells were counted to accurately estimate the number of cells spiked into the blood. The average number of Ep‐CAM and CK‐8, CK‐18, CK‐19 positive cells on FACS Caliber™ was used to calculate the cells recovery.

The mononuclear cells (MNCs) for FCM were incubated with monoclonal antibodies: anti‐ CD45‐PerCP, anti‐EPCAM‐PE, anti‐CD44‐APC and anti‐CD24‐FITC. About 20 μl of each antibody was needed, and the condition was 4°C for 30 min away from light. Then, the MNCs were washed two times. Finally, cells were resuspended with PBS and analyzed on FACS CaliburTM (BD Bio) using Cell Quest software (BD Bio).
