Flow Cytometry Laboratory

Chromosome Preparation protocol



1. Block cells with 0.05 µg/ml colcemid for 4-16 hours depending on the rate of growth. After this time estimate the percentage of cells in mitosis by resuspending cells in a solution of propidium iodide (50 µg/ml in 0.1% Triton-X) and analysing on a benchtop flow cytometer. It should be possible to get 40-60% of cells in mitosis.

2. Harvest cells in an appropriate manner and spin at 100g for 10 minutes.

3. Resuspend cells in fresh medium and spin again for a further 10 minutes.

4. Discard the supernatant by inverting the tube and remove the last few drops with a tissue. Disaggregate the cell pellet by vortexing gently or by flicking the tube. Add 5ml of a hypotonic solution (75mM potassium chloride), mix gently and leave for 10-30 minutes at room temperature. This solution causes cells to swell, so monitor this process microscopically.

5. Dissolve 12mg of digitonin in 5ml of distilled water by heating on a hotplate. Allow the digitonin solution to cool and then add 1ml of Chromosome Isolation Buffer (CIB: 20mM NaCl, 80mM KCl, 15mM Tris-HCL, 0.5mM EGTA, 2mM EDTA, 0.15% w/v 2-mercaptoethanol, 0.2mM spermine, 0.5mM spermidine pH7.2 in autoclaved distilled water). Make up to 10ml with distilled water and adjust the pH to 7.2. Place on ice.

6. After swelling, centrifuge the cells and carefully remove the supernatant with a Pasteur Pipette and agitate the tube gently to disaggregate the cells. Add 10 times the volume of the cell pellet of cold digitonin solution in CIB and aspirate gently. Mix a small amount of the preparation with a drop of propidium iodide and view under a fluorescence microscope. If the chromosomes are not monodispersed, aspirate the preparation more vigorously or vortex gently.

7. The preparation may be left at 4°C for several weeks with little deterioration of the flow karyotype.

8. To 1ml of the chromosome suspension add 30µl Hoechst 33258 (100µg/ml in distilled water), mix immediately. Add 40µl 15mM MgCl2 and 50µl Chromomycin A3 (2mg/ml in ethanol). Mix and leave the sample in the dark at 4°C for 2 hours in the dark.

9. The chromosome profile can be improved if 100µl of 100mM sodium citrate and 100µl of 250mM sodium sulphite are added 15 minutes prior to running on the cytometer.

10. Run the sample on a dual laser cytometer: primary laser at UV (351.1 - 363.8nm) to excite the Hoechst and secondary laser at 457nm to excite the chromomycin. Collect Hoechst fluorescence between 390 and 480nm, and chromomycin fluorescence above 520nm.

Further details on this protocol can be obtained by mailing Clare Hughes at ICRF. Back to Home Page            Back to Chromosome Page