11 Specimen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
149
genomic hybridization ( CGH) (Kallioniemi et al. 1992; du Manoir et al. 1993;
Joos et al. 1993; Joos and Lichter 1997) and multicolor FISH (Speicheret al.
1996; Schröck et al. 1996; Lichter 1997).
Preparation of chromosomes from peripheral blood cells
Because of their easy accessability peripheral blood lymphocytes are most
commonly used for cytogenetic analyses of constitutional chromosomal
aberrations. In general, heparinized blood is cultured in the presence of
mitogens (e.g. phytohemagglutinin, pokeweed mitogen or concanavalin
A) to re-stimulate non -cycling lymphocytes. Cells are arrested in metaphase
by application of colcemid, then treated with a hypotonicmedium to increase the nuclear volume. Finally, cells are fixed in methanol/acetic
acid and spread on glass slides. The protocol given below can be used
for primary cells from normal or leukemic individuals as well as for lymphoblastoid cell lines. In the latter case, no stimulation by mitogens is
needed. It should be noted that the yield of metaphase chromosomes is often much lower when clinical samples are investigated.
Subprotocol 1
Cultivation of Blood Lymphocytes
Materials
• NH4-heparinized blood.
• Culture medium: RPMI 1640 supplemetned with fetal calf serum (10o/o),
L-glutamine (1 o/o), penicillin (1 o/o), streptomycin (1 o/o), phytohemagglutinin (PHA) (l.So/o in water).
Procedure
1. Add 0.5-1 ml heparinized blood to 10 ml culture medium.
2. Incubate at 37°C (optional: in an atmosphere of So/o C02) for 71 h. Carefully resuspend settled cells once per day.
3. Proceed as described in Subprotocol 2.
Cultivation of
lymphocytes
149
genomic hybridization ( CGH) (Kallioniemi et al. 1992; du Manoir et al. 1993;
Joos et al. 1993; Joos and Lichter 1997) and multicolor FISH (Speicheret al.
1996; Schröck et al. 1996; Lichter 1997).
Preparation of chromosomes from peripheral blood cells
Because of their easy accessability peripheral blood lymphocytes are most
commonly used for cytogenetic analyses of constitutional chromosomal
aberrations. In general, heparinized blood is cultured in the presence of
mitogens (e.g. phytohemagglutinin, pokeweed mitogen or concanavalin
A) to re-stimulate non -cycling lymphocytes. Cells are arrested in metaphase
by application of colcemid, then treated with a hypotonicmedium to increase the nuclear volume. Finally, cells are fixed in methanol/acetic
acid and spread on glass slides. The protocol given below can be used
for primary cells from normal or leukemic individuals as well as for lymphoblastoid cell lines. In the latter case, no stimulation by mitogens is
needed. It should be noted that the yield of metaphase chromosomes is often much lower when clinical samples are investigated.
Subprotocol 1
Cultivation of Blood Lymphocytes
Materials
• NH4-heparinized blood.
• Culture medium: RPMI 1640 supplemetned with fetal calf serum (10o/o),
L-glutamine (1 o/o), penicillin (1 o/o), streptomycin (1 o/o), phytohemagglutinin (PHA) (l.So/o in water).
Procedure
1. Add 0.5-1 ml heparinized blood to 10 ml culture medium.
2. Incubate at 37°C (optional: in an atmosphere of So/o C02) for 71 h. Carefully resuspend settled cells once per day.
3. Proceed as described in Subprotocol 2.
Cultivation of
lymphocytes