Source: Gallerani, G. et.al. Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH. J. Vis. Exp. (2017).
Circulating tumor cells (CTC) isolation and characterization from the blood of cancer patients is a type of non-invasive liquid biopsy and associated with relapse and disease progression. This video describes the technique of DNA fluorescence-in-situ-hybridization (DNA FISH) on CTCs captured on a medical wire, to study a specific gene of interest.
1. 3D DNA-FISH Day 2
CAUTION: Special attention must be paid to the high temperature of the instruments and humid chamber.
2. 3D DNA-FISH Day 3
3. 3D microscope observation and analysis
Figure 1: Functionalized wire, schematic arrangement of ALK break-apart probes, scheme of expected patterns of ALK rearrangement, and special holder. (a) Red boxes highlight the three main parts of the wire: functionalized tip, wire-stopper, and un-functionalized tip. The functionalized tip is the most delicate part of the wire and must not be touched during handling to avoid cell detachment or damage. (b) The green and red probes each respectively bind to sequences upstream and downstream of the loci of ALK gene (on the left). On the right, exemplificative patterns of ALK arrangements are shown. Red and green co-localization signals on normal cells; separated green and red signals indicate an ALK gene chromosome break (translocation of ALK gene) and one green-orange colocalization signal (aberrant cell-1). One red signal and two green signals indicate the loss of one red signal, suggesting a chromosomal translocation and a deletion (aberrant cell-2). (c) Wire holder; red boxes highlight areas where care is needed when handling the wire during microscopy analysis. The wire can undergo a 360 ° analysis by turning the rotator.
The authors have nothing to disclose.
Tween 20 | BIO RAD | # 1706531 | |
Vysis 20X SSC (66g) | Abbott Molecular Inc. | # 30-804850 | powder to be resuspended in distilled water, as recommended |
Water | MilliPore | ||
DAPI, FluoroPure grade | Invitrogen | # D21490 | |
ZEISS Fluorescent Microscope Axioskop | ZEISS |