Culture of Peripheral Blood Lymphocytes for Chromosome Analysis

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Application Notes

The blood cell karyotyping method was developed to provide information about chromosomal abnormalities. Lymphocyte cells do not normally undergo subsequent cell divisions. In the presence of a mitogen, lymphocytes 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.

Protocol

  1. Inoculate approximately 0.5ml of heparinized whole blood into a glass or plastic tube with 10ml of PB-MAX medium (GIBCO Cat. No. 12557).

  2. Incubate the culture at 37ºC in 5% CO2 atmosphere for 72 hours.

  3. Add 0.5ug/mL of KaryoMAX™ Colcemid Solution (GIBCO Cat. No. 15212 or 15210) to each culture tube.

  4. Incubate the culture for an additional 15-30 minutes.

  5. Transfer the culture to a centrifuge tube and spin at 500xg for 5 minutes.

  6. Remove the supernatant and re-suspend the cells in 5-10ml of hypotonic 0.075M KCl (GIBCO Cat. No. 10575-090).

  7. Incubate at 37ºC for 10-12 minutes.

  8. Spin at 500xg for 5 minutes.

  9. 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.

  10. Repeat steps 7 and 8.

  11. Spin at 500xg for 5 minutes.

  12. 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.

  13. At this stage, the preparation can be stained with Orecin or Giemsa. Giemsa banding has become the most widely used technique, and the most common method to obtain this staining is to treat slides with Trypsin-EDTA 10X (Cat. No. 15400-054).

References

  1.    ACT Laboratory Procedure Manual, 1980, section 2, pgs.70-77 and 2nd edition, 1991, chapter 2 pg 24-30.

  2.    Barch, M.
LT075   16-Mar-2009