Culture of Bone Marrow and Peripheral Blood Cells for Chromosome Analysis
The hematopoietic cell karyotyping method was developed to provide information about chromosomal abnormalities. In the presence of a conditioned medium, acute and chronic nonlymphocytic leukemic cells in bone marrow and peripheral blood cultures are stimulated to enter into mitosis by DNA replication. After 48-72 hours, a mitotic inhibitor is added to the culture to stop mitosis in the metaphase stage. After treatment by hypotonic solution, fixation and staining, chromosomes can be microscopically observed and evaluated for abnormalities.
1. Inoculate approximately 0.5ml of bone marrow suspension or 0.5-1x107 Ficoll-separated peripheral blood cells into a plastic tube or tissue culture plate with 10ml of medium. Invert tubes gently to mix specimen.
2. Incubate the culture for 72-120 hours.
3. Add 0.1-0.2ml of Colcemid Solution (Cat. No. 12-004- 1) to each culture tube. Incubate the culture for an additional 15-30 minutes.
4. Transfer the culture to a centrifuge tube and spin at 500g for 5 minutes.
5. Remove the supernatant and re-suspend the cells in 5-10ml of hypotonic 0.075M KCl (Cat. No. 12-005-1). Incubate at 37ºC for 10-12 minutes.
6. Spin at 500g for 5 minutes.
7. Remove the supernatant, agitate the cellular sediment and add drop-by-drop 5-10ml of fresh, ice-cold fixative made up of 1 part acetic acid to 3 parts methanol. Leave in 4ºC for 10 minutes.
8. Repeat steps 6 and 7.
9. Spin at 500g for 5 minutes.
10. Re-suspend the cell pellet in a small volume 0.5-1ml of fresh fixative, drop onto a clean slide and allow to air dry.
11. At this stage, the preparation can be stained with Orecin or Giemsa. Giemsa banding has become the most widely used technique. The most common method to obtain this staining is to treat slides with Trypsin-EDTA 10X (Cat. No. 03-051-5).
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