Myeloid Leukemia

Chimerism Analysis 281
14. 310 Genetic Analyzer Capillaries 47 cm × 50 µm or 3100-Avant Capillary Array
36 cm (Applied Biosystems).
2.4. FISH Analysis of X and Y Chromosomes for Chimerism Analysis
in the Sex-Mismatched Transplant Setting
1. Eppendorf centrifuge.
2. Coated slides with 12 chambers (Roth Lactan).
3. Cover slips for individual chambers (see Note 2).
4. Glass jars for incubation of slides.
5. Heating plate.
6. Humid chamber (see Note 3).
7. Incubator (37°C).
8. Water bath (72°C).
9. Glass cover slips: 24 × 60 mm.
10. Fluorescence microscope equipped with appropriate filters for Spectrum Green,
Spectrum Orange, and DAPI.
11. Ethanol 70%, 85%, 96%.
12. 1X Phosphate-buffered saline (PBS).
13. Pepsin (Sigma).
14. Pepsin solution: 50 µg/mL in 0.01 N HCl; freshly prepared, prewarmed at 37°C.
15. Formaldehyde fixative: 1% formaldehyde, 1X PBS, 50 mM MgCl 2 (store at 4°C
in darkness).
16. Deionized water.
17. X and Y chromosome-specific DNA probe solution: CEP X spectrum orange/Y
spectrum green DNA Probe Kit (# 7J2050, VYSIS).
18. Rubber cement (Marabu).
19. 0.25X sodium saline citrate (SSC).
20. Tween-20 (Pharmacia).
21. Washing solution : 0.4X SSC, 0.2% Tween-20.
22. Vectashield mounting medium with DAPI (H-1200, Vector).
23. Immersion oil for fluorescence microscopy.
3. Methods
3.1. Isolation of Cell Subsets by Flow-Sorting
1. Starting material is peripheral blood (PB), usually anticoagulated with EDTA, or
bone marrow (BM), usually anticoagulated with heparin.
2. Determine the white blood cell count (WBC) using any available equipment and
determine the PB/BM volume required for the following steps (see Note 4).
3. Transfer the appropriate volume (containing 4 × 10 6 cells) to a 50-mL tube and
add 10 vol of RBC lysis buffer.
4. Incubate in a refrigerator at 4°C until lysis is complete (5–10 min).
5. Centrifuge for 10 min at 400g in a refrigerated centrifuge at 4°C.
6. Remove the supernatant and resuspend the pellet in 10 mL PBS.