Preparation of Metaphase Spreads

3.1.1. Preparation From Whole Peripheral Blood Cells

1. Mix 5 mL of whole blood with 5 mL of 1X PBS.

2. Provide 7.5 mL of separation medium and overlay carefully with whole blood/ 1X PBS mixture and centrifuge for 30 min at 950g.

3. With a Pasteur pipet, remove carefully the portion containing the interphase lymphocytes (white ring) and wash with 50 mL of 1X PBS.

4. Centrifuge for 10 min at 400g and remove the supernatant.

5. Suspend the cells in 5 mL of karyotyping medium (approx 106 cells/mL medium).

3.1.2. Preparation From Cultured Cells

These steps describe the treatment of adherent cells. Cells grow as suspension does not need the trypsin/EDTA treatment. In this case the method is aimed for 10 mL of cell suspension.

1. Remove the medium from a 75-cm2 culture flask.

3. Incubate at 37°C with 1 mL of trypsin-EDTA solution.

4. Add 9 mL of medium, harvest the solution, and centrifuge at 150gfor 10 min.

5. Deplete the supernatant and wash the pellet with 1X PBS.

6. Resuspend cells in karyotyping medium at concentration of 106 cells/mL medium.

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