Circulating Tumor Cell Isolation Protocol

For each 3.75 mL of blood, red blood cell (RBC) lysis was first performed using 1× Pharm Lyse solution (BD Biosciences). The RBC-depleted sample was then incubated with 4 tests of FITC-anti-CD45 (eBiosciences; clone HI30) for 30 min at 4 °C, followed by incubation with anti- EpCAM magnetic beads (Illumina)^{16 (link)} for 30 min at 4 °C. PE-anti-EpCAM (BD Biosciences; clone EBA-1) was then added for 30 min at 4 °C prior to immunomagnetic isolation using the Illumina MagSweeper^{16 (link)}.

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Lohr J.G., Adalsteinsson V.A., Cibulskis K., Choudhury A.D., Rosenberg M., Cruz-Gordillo P., Francis J., Zhang C.Z., Shalek A.K., Satija R., Trombetta J.T., Lu D., Tallapragada N., Tahirova N., Kim S., Blumenstiel B., Sougnez C., Lowe A., Wong B., Auclair D., Van Allen E.M., Nakabayashi M., Lis R.T., Lee G.S., Li T., Chabot M.S., Ly A., Taplin M.E., Clancy T.E., Loda M., Regev A., Meyerson M., Hahn W.C., Kantoff P.W., Golub T.R., Getz G., Boehm J.S, & Love J.C. (2014). Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer. Nature biotechnology, 32(5), 479-484.

Publication 2014

1× Pharm Lyse solution FITC-anti-CD45 anti- EpCAM magnetic beads PE-anti-EpCAM MagSweeper16

Blood Clone Epcam Fitc Isolation Red blood cell

Corresponding Organization :

Other organizations : Dana-Farber Cancer Institute, Broad Institute, Harvard University, Massachusetts Institute of Technology, Brigham and Women's Hospital, Massachusetts General Hospital, Dana-Farber Brigham Cancer Center, Howard Hughes Medical Institute, Ragon Institute of MGH, MIT and Harvard

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Variable analysis

independent variables

RBC lysis using 1× Pharm Lyse solution (BD Biosciences)
Incubation with FITC-anti-CD45 (eBiosciences; clone HI30) for 30 min at 4 °C
Incubation with anti-EpCAM magnetic beads (Illumina) for 30 min at 4 °C
Addition of PE-anti-EpCAM (BD Biosciences; clone EBA-1) for 30 min at 4 °C
Immunomagnetic isolation using the Illumina MagSweeper

dependent variables

Outcome of the immunomagnetic isolation process

control variables

Volume of blood (3.75 mL)
Temperature (4 °C)
Incubation time (30 min)

controls

No positive or negative controls were explicitly mentioned.

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