Isolation and Analysis of Circulating Tumor Cells

Whole blood drawn from healthy donors was collected in heparin-treated tubes and kept at ambient temperature. Studies using human blood were reviewed and approved by UIC institutional review board (IRB) (protocols #2012–0139 for tumor cell-spiked blood specimens and #2013–1033 for clinical PCa patient blood specimens). Fluorescence-labeled BCa or PCa cells were spiked into 3 mL of whole blood at a final concentration of 1 × 10⁵ tumor cells/mL blood. Mononuclear cells including tumor cells in buffy coat were separated from whole blood using Ficoll-Paque Plus (Stemcell Technologies Inc., Vancouver, Canada) as described in our previous publication.^{21 (link)} After washing the buffy coat twice with the 2% FBS-containing PBS, the recovered cells were suspended in 3 mL of the complete cell culture media and used for subsequent experiments. For the clinical samples, 12 mL of the blood specimen obtained from prostate cancer patients were used for separation and resuspended with 12 mL of complete media. The experiments were performed within 48 hrs after blood drawing.