Myeloid Leukemia

286 Lion and Watzinger
Fig. 3. Detection of male and female cells by fluorescence in situ hybridization. In
the sex-mismatched transplantation setting, cells of donor and recipient origin can be
detected by fluorescence in situ hybridization (FISH) based on the use of probes specific
for the X and Y chromosomes. The probes are labeled with different dyes, e.g.,
green for the X chromosome and red for the Y chromosome, as shown in this example.
In interphase nuclei, female cells (arrow) display two green signals (XX), while the
remaining six male cells show one green and one red signal (XY). This technique
permits accurate quantification of donor and recipient derived hematopoietic cells.
3.4.2. Pretreatment of Cells
1. Perform fixation of the air-dried slide(s) by passing through an ethanol series
(70%, 85%, 96%), each step for 3 min, then air-dry slide(s) at RT.
2. Permeabilize the cells by incubating the slide(s) in pepsin solution for 20 min at
37°C.
3. Transfer the slide(s) to 1X PBS for 5 min at room temperature.
4. Incubate the slide(s) in the formaldehyde fixative for 10 min at room temperature
(in darkness).
5. Transfer the slide(s) to 1X PBS for 5 min at room temperature.
6. Briefly rinse the slide(s) with deionized water.
7. Dehydrate the cells by incubating in an ethanol series (70%, 85%, 96%), each
step for 3 min, then air-dry the slide(s) at room temperature.
3.4.3. Hybridization
1. Apply 0.6 µL of the DNA probe solution to each chamber.
2. Cover each chamber with a fitting cover slip (avoid bubble formation).
3. Seal each slip with rubber cement.