11 Specirnen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
153
Procedure
1. Add colcemid solution to adherently growing cells (final concentration
of 0.1 J..Lg/ml). Incubate for 2 h at 37°C.
2. Suspend mitotic cells by strong shaking (tlasks can be knocked on
bench); monitor by microscopy; when mitotic cells are in suspension,
proceed with step 4.
3. If cells remain adherent to the surface, discard culture medium and add
trypsin-EDTA-solution to cover the cells. Incubate for about 10-20 min,
then stop the digestion by adding medium (5-1 0 tim es the volume of the
trypsin-EDTA-solution) or FCS (same volume as the trypsin-EDTA-solution).
4. Transfer cell suspension into a 50 ml tube. Spin for 15 min at 150g.
5. Remave supernatant and proceed as described in Subprotocol2 (step 5).
Subprotocol 5
Treatment of Blood Smears
Interphase cytogenetics using FISH can be performed on routine blood
smears that have been previously Wright's stained for hematological analysis (see Figure 1). Thus, cell morphology can be related to specific chromosomal aberrations, allowing the detection of celllineages involved in tumorigenesis (see Anastasi et al. 1992; Bentz et al. 1993b; Stilgenbauer et al.
1992). The protocol presented below has been successfully used on previously stained blood smears that have been stored for up to one year at
room temperature in the clinical hematology laboratory.
Materials
• Phosphate-buffered saline (PBS).
• TE: 10mM Tris-HCl, pH 7.5; 1 mM EDTA
• Methanol/acetic acid (19:1)
• Methanol/acetic acid (3:1)
• 70%, 90%, 100% ethanol.
Preparation
of fibroblasts
153
Procedure
1. Add colcemid solution to adherently growing cells (final concentration
of 0.1 J..Lg/ml). Incubate for 2 h at 37°C.
2. Suspend mitotic cells by strong shaking (tlasks can be knocked on
bench); monitor by microscopy; when mitotic cells are in suspension,
proceed with step 4.
3. If cells remain adherent to the surface, discard culture medium and add
trypsin-EDTA-solution to cover the cells. Incubate for about 10-20 min,
then stop the digestion by adding medium (5-1 0 tim es the volume of the
trypsin-EDTA-solution) or FCS (same volume as the trypsin-EDTA-solution).
4. Transfer cell suspension into a 50 ml tube. Spin for 15 min at 150g.
5. Remave supernatant and proceed as described in Subprotocol2 (step 5).
Subprotocol 5
Treatment of Blood Smears
Interphase cytogenetics using FISH can be performed on routine blood
smears that have been previously Wright's stained for hematological analysis (see Figure 1). Thus, cell morphology can be related to specific chromosomal aberrations, allowing the detection of celllineages involved in tumorigenesis (see Anastasi et al. 1992; Bentz et al. 1993b; Stilgenbauer et al.
1992). The protocol presented below has been successfully used on previously stained blood smears that have been stored for up to one year at
room temperature in the clinical hematology laboratory.
Materials
• Phosphate-buffered saline (PBS).
• TE: 10mM Tris-HCl, pH 7.5; 1 mM EDTA
• Methanol/acetic acid (19:1)
• Methanol/acetic acid (3:1)
• 70%, 90%, 100% ethanol.
Preparation
of fibroblasts