Cellsearch kit

The blood sample collection from stage III–IV breast cancer patients was permitted by the Institutional Review Board at Northwestern University and complied with NIH guidelines for human subject studies. Blood samples were collected in collected in CellSave preservative tubes for CellSearch platform analyses and in EDTA tubes for flow cytometry analyses. CellSearch kit and anti-ICAM1 antibody (conjugated to PE, BD# 555511) were used to enrich CTCs for immunofluorescence staining. Live blood cell samples were centrifuged and after red blood cell lysis (lysis buffer Sigma cat# R7757), white blood cells were stained with antibodies for lineage markers such as CD45 (leukocytes), EpCAM (epithelial), and candidate markers ICAM1 and CD44 for flow cytometry analysis on FACS LSR (BD Biosciences). Single and clustered tumor cells were gated for ICAM1 expression (%). In some sample processing, CD45⁺ PBMCs were depleted using the kit (Miltenyi Biotec Depletion column cat#130-042-901).

Blood samples from breast cancer patients were collected in EDTA tubles for flow cytometry analyses of live cells or collected in CellSave preservative tubes for CellSearch platform analyses. Live cells were centrifuged and resuspended in red blood cell lysis buffer to remove red blood cells. The remaining cells were washed and stained with antibodies for different makers such as EpCAM for epithelial cells, CD45 for leukocytes, and candidate markers such as EGFR, and then analyzed through the FACS LSR (BD Biosciences) instrument. Single and clustered EpCAM⁺ tumor cells were gated for EGFR expression (%). Two sample paired t-test were used to compare the proportion of EGFR⁺ tumor cells within the singlets and clustered CTCs. CellSearch kit and anti-EGFR antibody (conjugated to PE, BD Bioscience cat# 555997) were used to enrich CTCs for immunofluorescence staining.