Journal: Micromachines

Article Title: An Immune–Magnetophoretic Device for the Selective and Precise Enrichment of Circulating Tumor Cells from Whole Blood

doi: 10.3390/mi11060560

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: 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

Journal: Micromachines

Article Title: An Immune–Magnetophoretic Device for the Selective and Precise Enrichment of Circulating Tumor Cells from Whole Blood

doi: 10.3390/mi11060560

Figure Lengend Snippet: Isolation and characterization of CTCs from a cholangiocarcinoma patient: ( A ) Immunofluorescence image showing EPCAM-positive cells isolated from a cholangiocarcinoma patient. EPCAM-positive cells were immunostained with DAPI for nuclear staining (blue), an anti-PDL1 antibody (Alexa flour 488; Green), and an anti-Pan CK antibody (Alexa flour 594; Orange red) to detect CTCs; an anti-CD45 antibody was used for leukocytes (Cy3; Yellow). ( B ) Vimentin-positive cells isolated from a cholangiocarcinoma patient were immunostained with DAPI for nuclear staining (blue), an anti-PDL1 antibody (Alexa flour 488; Green), and an anti-Pan CK antibody (Alexa flour 594; Orange red) to detect CTCs; an anti-CD45 antibody was used for leukocytes (Cy3; Yellow). ( C ) MET-positive cells isolated from a cholangiocarcinoma patient were immunostained with DAPI for nuclear staining (blue), an anti-PDL1 antibody (Alexa flour 488; Green), and an anti-Pan CK antibody (Alexa flour 594; Orange red) to detect CTCs; an anti-CD45 antibody was used for leukocytes (Cy3; Yellow). The yellow arrow indicates CK+/PDL1+ cells. All microscopic images were obtained using a Nikon Eclipse Ni microscope equipped with an Infinity3 camera. ( D ) Graph showing the difference in CTC counts determined using EPCAM-, vimentin- or ME-based methods. The number of PDL1-positive CTCs observed in each case is also shown.

Article Snippet: 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, Immunofluorescence, Staining, Microscopy

Journal: Gut

Article Title: CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism

doi: 10.1136/gutjnl-2014-308325

Figure Lengend Snippet: Hepatic transforming growth factor-β (TGF-β) is reduced in CD248-deficient mice following carbon tetrachloride (CCl 4 )-induced injury. CD248 −/− mice and wild-type (WT) mice were injected biweekly with CCl 4 for 8 weeks (n=6). (A) Transcript levels of growth factors and (B) enzymes involved in matrix remodelling were analysed by qPCR of whole liver tissue. (C) Representative images of livers from untreated CD248 −/− mice and WT mice stained with vimentin or desmin are shown. Scale bar=100 μm. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HGF, hepatocyte growth factor; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TIMP, tissue inhibitor of metalloproteinase.

Article Snippet: For human sections and primary human HSCs, the following antibodies were used: monoclonal mouse IgG1 antihuman CD248 (generated in-house), monoclonal mouse IgG2a antihuman α-SMA (Dako, Ely, UK), monoclonal mouse IgG1 antihuman glial fibrillary acidic protein (GFAP) (Dako), monoclonal mouse IgG1 antihuman vimentin (Vector Laboratories) and monoclonal mouse IgG1 antihuman CD90 (eBioscience).

Techniques: Injection, Staining, Derivative Assay