150
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
ln advance
Preparation of
chromosomes and
nuclei
Subprotocol 2
Preparation of Metaphase Chromosomes and Interphase Nuclei from
Blood lymphocytes
Materials
• Colcemid solution: prepare stock solution of 10 J..Lg/ml in water and store
at -20°C.
• Hypotonicsolution (per 1): 3g KCl; 0.2 g EGTA; 4.8 g HEPES; adjust pH to
7 with 1 M KOH.
• Fixative solution: 3 parts methanol (abs.), 1 part acetic acid (glacial).
Procedure
Pre-cleaned microscopic slides are washed in ethanol for several hours,
dipped several times in water and dried immediatedly before cells are
dropped onto them.
1. Following incubation of blood cells in culture medium for 71 h add
colcemid stock solution to a final concentration of 0.1 J..Lg/ml.
2. Incubate for 10-60 min at 37°C (incubation time should be optimized
for different cell sources).
3. Aliquot into two 15 ml tubes (polystyrene) and centrifuge at 200 g for 10
min in a clinical centrifuge.
4. Remove supernatant, resuspend cell pellet in remaining medium, and
add a few drops of hypotonic solution.
5. Add 5 ml of hypotonic solution to each tube, resuspend and incubate
for 15 min at 37°C.
6. Spinat 200 g for 10 min.
7. Remove supernatant, resuspend the pellet, add a few drops of ice-cold
fixative solution.
8. Slowly (!) add 5 ml of cold fixative solution to each tube while constantly
whirling the suspension.
9. Centrifuge at 200 g in a cooled (4°C) centrifuge.
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
ln advance
Preparation of
chromosomes and
nuclei
Subprotocol 2
Preparation of Metaphase Chromosomes and Interphase Nuclei from
Blood lymphocytes
Materials
• Colcemid solution: prepare stock solution of 10 J..Lg/ml in water and store
at -20°C.
• Hypotonicsolution (per 1): 3g KCl; 0.2 g EGTA; 4.8 g HEPES; adjust pH to
7 with 1 M KOH.
• Fixative solution: 3 parts methanol (abs.), 1 part acetic acid (glacial).
Procedure
Pre-cleaned microscopic slides are washed in ethanol for several hours,
dipped several times in water and dried immediatedly before cells are
dropped onto them.
1. Following incubation of blood cells in culture medium for 71 h add
colcemid stock solution to a final concentration of 0.1 J..Lg/ml.
2. Incubate for 10-60 min at 37°C (incubation time should be optimized
for different cell sources).
3. Aliquot into two 15 ml tubes (polystyrene) and centrifuge at 200 g for 10
min in a clinical centrifuge.
4. Remove supernatant, resuspend cell pellet in remaining medium, and
add a few drops of hypotonic solution.
5. Add 5 ml of hypotonic solution to each tube, resuspend and incubate
for 15 min at 37°C.
6. Spinat 200 g for 10 min.
7. Remove supernatant, resuspend the pellet, add a few drops of ice-cold
fixative solution.
8. Slowly (!) add 5 ml of cold fixative solution to each tube while constantly
whirling the suspension.
9. Centrifuge at 200 g in a cooled (4°C) centrifuge.