LSC Application: FISH, fluorescence in situ hybridization, correlated to DNA content

The LSC can perform automatic counting of fluorescence-in-situ-hybridization probe spots with the simultaneous measurement of cellular DNA.
WinCyte™ software automatically searches for probe spots within nuclear boundaries during the scanning process. Probe spot area, total probe fluorescence, maximum pixel fluorescence, and the number of probe spots per cell are recorded along with other cell data. The LSC can scan sufficient numbers of cells in order to perform statistical analysis of DNA content and determine the characteristics of well-defined probe spots in order to eliminate overlapping spots from the distribution.

Cells were selected by setting a single cell gate (A in Window 2) based on PI fluorescence and cell area. Single cells were directed to a dot plot of probe area vs. probe Max Pixel (Window 3) in order to select non-overlapping probe spots (B) from overlapping spots (C). The corresponding probe spot count histograms are shown in Windows 4 - 6.

in situ hybridization probe spots correlated to DNA
content

Visualization of X-chromosome probe spot count by CompuSort using laser scan images provides easy verification of the gating algorithms.

x-chromosome probe spots

Sample Preparation
Cytospin preparations of human lymphocytes or cell lines cultured directly in chamber slides (Becton Dickinson Labware, Franklin Lakes, NJ) were prepared for LSC analysis by fluorescence in situ hybridization using a modified technique of Weigant and Raap (Current Protocols in Cytometry, 8.2.1, Wiley, 1997) using a biotinylated X-chromosome probe (Oncor, Gaithersburg, MD) and counterstained with propidium iodide (PI, 0.1-0.5 µg/ml depending upon the specimen) for cellular DNA content and avidin FITC for FISH fluorescence. In some preparations, FISH probes were amplified by staining with a murine anti-FITC antibody (DAK 4, Dako Corp., Carpenteria, CA) followed by GAM-FITC (Dako Corp., Carpenteria, CA), with PBS washes in between. Slide preparations were coverslipped with a 50% glycerol/PBS solution and stored in the dark prior to analysis.
LSC Analysis
Slides were placed on the LSC stage, and an area was selected for scanning with the 40X objective using green epi-fluorescence to visualize cells without fading the FISH spots. The argon laser was used with 5 mW laser power. Cells were contoured based upon a PI fluorescence threshold of 600 fluorescence units above background and a minimum cell area of 100 pixels. FISH X-chromosome probes were detected by contouring on FITC fluorescence and a minimum of 20 pixels in fluorescence area.