3 FISH on Chromosome Preparations of Peripheral Blood
75
goxigenated probe with anti-digoxigenin-rhodamine leads to red signals. After counterstaining of the nuclei with DAPI and addition of
antifade solution, slides can be evaluated on a fluorescence microscope.
Procedure
Preparation of peripheral blood lymphocytes suspension
1. Add 1 ml of heparinized blood to 9 ml of cell culture medium (e.g.
RPMI 1640 mediuml 20% fetal calf seruml 300 f..lg/ml L-glutaminel
1 D/ml penicillin/l f..lglml streptomycinl 0.1 ml phytohaemagglutinine
{Gibco BRL, 061-00576C}), mix the suspension carefully and incubate
for 72 h at 37°C/5%CO z (see comments 2 and 3).
2. 16 h before harvesting, add 180 f..lg of BrdU to the 10 ml of the cell
culture (comment 4).
3. 30 min before harvesting of the cells 1 f..lg of colcemid is added to a 10
ml culture flask, the flask is mixed gently and incubated at 37°CI
5%CO z •
4. The fluid is put into a 15-ml tube - sterile conditions no longer need to
be observed.
5. Centrifuge the solution at room temperature (= RT) for 8 min at 1000
rpm and discard the supernatant by sucking it off carefully with a glass
pipette (1 ml of supernatant is left in the tube to avoid loss of material).
6. For hypotonic treatment the pellet is resuspended in 10 ml 0.075 M KCI
(37°C) and incubated at 37°C for 20 min.
7. Slowly add 0.6 ml of Carnoy's fIxative (= methanol/glacial acetic acid
3:1) (4°C) and mix the solution carefully.
8. Repeat step 5.
9. Resuspend the pellet in 10 ml of fIxative (4°C) and incubate at 4°C for
20 min.
10. Repeat step 5.
11. Resuspend the pellet in 5 ml of fIxative (4°C) and repeat step 5.
12. Repeat step 11 twice.
75
goxigenated probe with anti-digoxigenin-rhodamine leads to red signals. After counterstaining of the nuclei with DAPI and addition of
antifade solution, slides can be evaluated on a fluorescence microscope.
Procedure
Preparation of peripheral blood lymphocytes suspension
1. Add 1 ml of heparinized blood to 9 ml of cell culture medium (e.g.
RPMI 1640 mediuml 20% fetal calf seruml 300 f..lg/ml L-glutaminel
1 D/ml penicillin/l f..lglml streptomycinl 0.1 ml phytohaemagglutinine
{Gibco BRL, 061-00576C}), mix the suspension carefully and incubate
for 72 h at 37°C/5%CO z (see comments 2 and 3).
2. 16 h before harvesting, add 180 f..lg of BrdU to the 10 ml of the cell
culture (comment 4).
3. 30 min before harvesting of the cells 1 f..lg of colcemid is added to a 10
ml culture flask, the flask is mixed gently and incubated at 37°CI
5%CO z •
4. The fluid is put into a 15-ml tube - sterile conditions no longer need to
be observed.
5. Centrifuge the solution at room temperature (= RT) for 8 min at 1000
rpm and discard the supernatant by sucking it off carefully with a glass
pipette (1 ml of supernatant is left in the tube to avoid loss of material).
6. For hypotonic treatment the pellet is resuspended in 10 ml 0.075 M KCI
(37°C) and incubated at 37°C for 20 min.
7. Slowly add 0.6 ml of Carnoy's fIxative (= methanol/glacial acetic acid
3:1) (4°C) and mix the solution carefully.
8. Repeat step 5.
9. Resuspend the pellet in 10 ml of fIxative (4°C) and incubate at 4°C for
20 min.
10. Repeat step 5.
11. Resuspend the pellet in 5 ml of fIxative (4°C) and repeat step 5.
12. Repeat step 11 twice.