Flow cytometry is a technology which can not only give information of high statistical precision and subpopulation quantification but also analyze cells NVP-AUY922 individually and rapidly, compared with immunocytochemistry [14, 15] and reverse transcriptase polymerase chain reaction (RT-PCR) [16, 17]. In this study, flow cytometry was used to detect occult tumor cells in peripheral blood of patients with breast cancer. The detection of CTCs in peripheral blood of 48 patients was intended to find the relationship of CK19+ cell percentage with disease

progress. CK19 was positive in the peripheral white blood cells of breast cancer patients at stages II to IV, but not the patients at stage I and healthy controls. The percentage of CK19+ cells was increased following the severity of the disease and decreased Alpelisib in vivo after lumpectomy and chemotherapy. Methods Cell line The A431 (human epithelial carcinoma) cell line obtained from the American Type Culture Collection was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal calf serum (both from GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% CO2. Subculture was performed when confluence reached 70%. Patients Breast cancer patients were treated at the Affiliated Hospital of Anhui Medical University. The cohort included 7 patients with benign tumor, 34 patients

with primary breast cancer and 7 patients with metastatic breast cancer from October 2006 to April 2008. The patients underwent lumpectomy except those with distant metastases. And we detected CK19 expression of 15 patients

with primary breast cancer during three month chemotherapy. Blood samples were obtained with informed consent after approval of the protocol by the Ethics Committee of the University of Science and Technology of China. Control blood samples were collected from 25 healthy female volunteers. Blood sample preparation The first 8 ml of blood was discarded to avoid epithelial contamination before the collection of 5 ml blood sample. Human white blood cells were isolated from adult peripheral blood using RBC lysis buffer (RX-2-1-2 U-gene China). Briefly, 3 ml blood and 15 ml RBC lysis buffer were mixed with vortex and kept on ice for 15 min until Fossariinae pellucid, then were centrifuged at 450 g for 10 min. Cells were suspended with 5 ml RBC lysis buffer and centrifuged at 450 g for 10 min again followed by twice rinse with PBS. Immunofluorescence staining A431 cells were counted onto glass slides at a concentration of 5 × 105 cells per spot. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, rinsed in PBS, and incubated with FITC-conjugated mouse anti-human CK19. Laser scanning confocal microscopy was performed and the data were processed with MetaMorph program. Flow cytometric analysis After fixation with 1% paraformaldehyde for 1 hour at room temperature, A431 cells or leukocytes were permeabilized with 0.