Article Title: An Immune–Magnetophoretic Device for the Selective and Precise Enrichment of Circulating Tumor Cells from Whole Blood
Figure Lengend Snippet: Isolation and enumeration of CTCs from non-small cell lung cancer (NSCLC) patients: ( A ) the detailed GenoCTC workflow using clinical samples is summarized as follows: (1) Whole blood is drawn from cancer patients. (2) The biotinylated anti-EPCAM antibody is conjugated with 1 μm streptavidin-coated magnetic nanobeads. (3) Anti-EPCAM antibody-coated beads and whole blood are incubated together, and the nanobeads will specifically bind onto EPCAM-positive cells. (4) EPCAM-positive cells are isolated using GenoCTC with a disposable GenoChip. (5) Molecular analysis is conducted in various ways using isolated EPCAM-positive cells. ( B ) Representative fluorescent images of isolated EPCAM-positive cells from three NSCLC patients are shown. Cells were immunostained with DAPI for nuclear staining (blue), an anti-cytokeratin 18 (CK18) antibody for CTCs (Alexa flour 488; Green), and an anti-CD45 antibody for leukocytes (Alexa 594; red). ( C ) EPCAM-positive cells isolated from the whole blood of 10 NSCLC patients were enumerated, and the number of cells observed varied between patients. EPCAM-positive cell counts ranged from 2 to 112 in 3.5 mL of whole blood, and the average count of the cells was 20.75, showing 95% confidence. The orange shaded area show the serial assessment of CTCs from those patients. The grey arrows indicate the clinical changes in the disease status determined by a physician. SD: stable disease, PR: partial response, PD: progressive disease. The orange arrows indicate the increase or decrease in CTC counts during the serial assessment.
Article Snippet: Preparation of Antibody-Coated Magnetic Microbeads Magnetic microbead-conjugated antibodies for CTC isolation were prepared in phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) by incubation of biotinylated anti-human EPCAM (eBioscience, Thermo Fisher Scientific, Inc.), biotinylated anti-human vimentin (R & D systems, Biotechne., Minneapolis, MN, USA), or biotinylated anti-human MET (eBioscience, Thermo Fisher Scientific, Inc.) with streptavidin-coated magnetic beads (Size 1 µm) (Dynabead, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature.
Techniques: Isolation, Incubation, Staining