29 Spectral Imaging Analysis of Metaphase and Interphase Cells
369
- Procedure
Metaphase spreads
Caution: Slide cleaning is an essential step prior to dropping the metaphase cells. To remove the most adherent residues, the glass surface
can be wiped with Kim wipes which have been soaked with ethanol or
fIxative. Watch the surface. When smears appear, it is time to change
the paper.
A. Metaphase spreads are made from short-term cultures of normal
male lymphocytes
(For this part, refer to Chap. 3, FISH on Chromosomes Preparations of
Peripheral Blood by Thomas Liehr and Uwe Claussen).
1. Cultivate normal male lymphocytes for 72 h at 37°C in RPMI 1640 supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin,
1% L-glutamine, 1.32% heparin, and 4% phytohemagglutinin.
2. Block cultures for 17 h with methotrexate (10- 5 M).
3. Incubate cultures in RPMI containing 1% thymidine (fmal concentration 10- 5 M) for 5 h.
4. Block cells in mitosis with a 10 min treatment with colcemid.
5. Harvest and incubate cells in 75 mM KCl for 20 min at 37°C.
6. Pellet the cells and fIx them in freshly made fIxative (methanol/acetic
acid, 3/1, vol/vol).
7. Drop cells on ethanol-cleaned slides inside a CDS-5 Cytogenetic Drying Chamber at 25°C and 45-50% relative humidity.
8. Check chromosome metaphase spreads using a phase contrast microscope. If there is any cytoplasmic residue visible around the metaphase
spread, wash the remaining cells with fIxative several times again before dropping them. Ideally, the chromosome metaphase spread appeared dark black. If metaphase spreads appear glassy, it suggests
the cells have dried too slowly. Decrease the chamber's humidity by
2-5%. If metaphase spreads appear gray, it suggests the cells have dried
too fast. Increase the chamber's humidity by 2-5%.
9. Store slides in slide boxes for at least two weeks at room temperature,
then place in sealed plastic bags fIlled with nitrogen gas at -20°C until
used.
369
- Procedure
Metaphase spreads
Caution: Slide cleaning is an essential step prior to dropping the metaphase cells. To remove the most adherent residues, the glass surface
can be wiped with Kim wipes which have been soaked with ethanol or
fIxative. Watch the surface. When smears appear, it is time to change
the paper.
A. Metaphase spreads are made from short-term cultures of normal
male lymphocytes
(For this part, refer to Chap. 3, FISH on Chromosomes Preparations of
Peripheral Blood by Thomas Liehr and Uwe Claussen).
1. Cultivate normal male lymphocytes for 72 h at 37°C in RPMI 1640 supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin,
1% L-glutamine, 1.32% heparin, and 4% phytohemagglutinin.
2. Block cultures for 17 h with methotrexate (10- 5 M).
3. Incubate cultures in RPMI containing 1% thymidine (fmal concentration 10- 5 M) for 5 h.
4. Block cells in mitosis with a 10 min treatment with colcemid.
5. Harvest and incubate cells in 75 mM KCl for 20 min at 37°C.
6. Pellet the cells and fIx them in freshly made fIxative (methanol/acetic
acid, 3/1, vol/vol).
7. Drop cells on ethanol-cleaned slides inside a CDS-5 Cytogenetic Drying Chamber at 25°C and 45-50% relative humidity.
8. Check chromosome metaphase spreads using a phase contrast microscope. If there is any cytoplasmic residue visible around the metaphase
spread, wash the remaining cells with fIxative several times again before dropping them. Ideally, the chromosome metaphase spread appeared dark black. If metaphase spreads appear glassy, it suggests
the cells have dried too slowly. Decrease the chamber's humidity by
2-5%. If metaphase spreads appear gray, it suggests the cells have dried
too fast. Increase the chamber's humidity by 2-5%.
9. Store slides in slide boxes for at least two weeks at room temperature,
then place in sealed plastic bags fIlled with nitrogen gas at -20°C until
used.