Isolation of Chromosomes for Flow Analysis and Sorting

I. INTRODUCTION
The bivariate analysis of mammalian chromosomes using the flow cytometer was first described by Gray and colleagues in 1979. In this method, metaphase chromosomes are released into a stabilising buffer and stained with two dyes, Hoechst 33258 (with specificity for AT-rich regions of DNA) and chromomycin A3 (specificity for GC-rich DNA) and then analysed or sorted using the flow cytometer. However, the technique has remained a somewhat specialist application limited to a relatively small number of laboratories. One reason for this is the requirement for the flow cytometer to be equipped with two large water-cooled and expensive lasers in order to excite the two dyes. However, arguably the most critical factor is the isolation of intact metaphase chromosomes from actively dividing cells. Several chromosome isolation procedures have been described (Sillar and Young 1981; van den Engh et al., 1984, 1985, 1988; general reference, Gray, 1989). Of these procedures, methods based on the stabilisation of chromosomes with polyamines (Sillar and Young, 1981) have proved to be most reliable for not only flow karyotype resolution, but also for the generation of high molecular weight DNA from sorted chromosomes. This article describes the procedure for isolating chromosomes from human lymphoblastoid cell lines using a polyamine buffer.

II. MATERIALS
Spermine tetrahydrochloride (Cat. No. S2876), spermidine trihydrochloride (Cat. No. S2501), Hoechst 33258 (Cat. No. B2883), chromomycin A3 (Cat. No. C2659), gentian violet (Cat. No. G2039), propidium iodide (Cat. No. P4170), Triton X-100 (Cat. No. X100), Trizma base (Tris-base, Cat. No. T6791), EGTA (Cat. No. E3889), EDTA (Cat. No. E5134), potassium chloride (Cat. No. P9541), sodium chloride (Cat. No. S3014), sodium sulphite (Cat. No. S8018), trisodium citrate (Cat. No. C3434), RPMI1640 medium (Cat. No. R8758), L-glutamine /penicillin/streptomycin solution (Cat. No. G6784), dithiothreitol (Cat. No. D9779), and magnesium sulphate (Cat. No. M2773) are obtained from Sigma. Foetal bovine serum (Cat. No. 16000-044) is obtained from Gibco. Invitrogen Corp.

III. PROCEDURES
A. Tissue Culture and Chromosome Preparation
Solutions

1. Culture medium: To 500 ml of RPMI 1640 medium add 100ml of foetal bovine serum and 5ml of L-glutamine/penicillin/streptomycin additive.
2. Polyamine buffer and stain stock solutions
   1. Dissolve 1.18 g of Tris-base and 832mg of EDTA in sterile distilled water, adjust pH to 8.0, and bring final volume to 100ml.
   2. Dissolve 1.18 g of Tris-base and 190 mg of EGTA in sterile distilled water, adjust pH to 8.0, and bring final volume to 100ml.
   3. Dissolve 5.96 g of potassium chloride in sterile distilled water and bring final volume to 100ml.
   4. Dissolve 1.17g of sodium chloride in sterile distilled water and bring final volume to 100ml.
   
   Solutions should be sterile filtered and stored at 4-10°C for up to 1 month.
   
   
   5. Dissolve 1.39g of spermine tetrahydrochloride in sterile distilled water and bring final volume to 10ml. Aliquot and store at -20°C.
   6. Dissolve 2.55g of spermidine trihydrochloride in sterile distilled water and bring final volume to 10ml. Aliquot and store at -20°C.
   7. Dissolve 1 g of dithiothreitol in sterile distilled water and bring final volume to 21.6 ml. Sterile filter and store at 4-10°C.
   8. Dissolve 3.15g of sodium sulphite in sterile distilled water and bring final volume to 100 ml. Do not store.
   9. Dissolve 2.94g of trisodium citrate in sterile distilled water and bring final volume to 100 ml. Do not store.
   10. Dissolve 10mg of Hoechst 33258 in sterile distilled water and bring final volume to 10ml. Sterile filter and store in the dark at 4-10°C.
   11. Dissolve 5 mg of chromomycin A3 in ethanol and bring final volume to 2.5 ml. Aliquot and store at -20°C.
   12. Dissolve 1 mg of propidium iodide in sterile distilled water and bring the final volume to 10ml.
   13. Dissolve 1.2g of magnesium sulphate in sterile distilled water and bring final volume to 100ml. Sterile filter and store at 4-10°C.
   14. Add 1 ml of glacial acetic acid and 10mg of gentian violet to sterile distilled water and bring the final volume to 100ml (Turck's stain). Store at 4-10°C.
   
   
   

Steps

1. Grow 20ml of the lymphoblastoid cell line to near confluence and add a further 30 ml of medium to the flask, gently breaking up any cell clumps. Incubate for 24 h.
2. Add 0.5 ml of colcemid and mix gently. Incubate for 6h.
3. Transfer the cell suspension to a 50-ml centrifuge tube and centrifuge at 200g for 10 min. Decant the supernatant and place the tube, inverted, on an absorbent paper tissue.
4. Dilute 4.7 ml of stock 800 mM potassium chloride solution to 50ml with sterile distilled water and filter through a sterile 0.2-µm filter. Add 10ml to the cell pellet and resuspend gently. Incubate at room temperature for 15 min.
5. Monitor swelling by staining 10 µl of the cell suspension with an equal volume of Turck's stain and viewing in a haemocytometer using phase-contract microscopy. Monitor cell bursting by staining 20µl of the cell suspension with 1 µl of propidium iodide solution and viewing in a haemocytometer using fluorescence microscopy.
6. To make the polyamine buffer, mix together 10ml of each of the four stock solutions (a-d), 50µl of spermine tetrahydrochloride, 50µl spermidine trihydrochloride, 250 µl of Triton X-100, 500 µl of dithiothreitol, and 50ml of sterile distilled water, adjust pH to 7.2 with hydrochloric acid, and bring the final volume to 100 ml. Pass through a sterile 0.2-gm filter.
7. Centrifuge cell suspension at 400g for 10min, decant supernatant, and invert the tube on a paper tissue. Add 3ml of the polyamine buffer, resuspend gently, and incubate on ice for 10min.
8. Vortex the cell suspension for 10s at a speed that produces a swirling film of suspension around the wall of the tube.
9. Monitor the chromosome release by staining 20µl of the cell suspension with 1 µl of propidium iodide solution and viewing in a haemocytometer using fluorescence microscopy. Check that the majority of chromosomes are free in solution and not clumped. If clumps are apparent, vortex for periods of 5s until few clumps are present. Too vigorous or too long vortexing will increase the number of broken chromosomes and debris.
10. Centrifuge 850µl of the chromosome suspension at 100g for 1 min. Transfer 740µl of the supernatant to a tube suitable for use on the flow cytometer and add 20µl of chromomycin A3, mixing immediately.
11. Add 20 µl of magnesium sulphate. Dilute 5 µl of Hoechst with 45µl of sterile distilled water and add 20µl to the chromosome suspension and mix well. Incubate for at least 2h on ice.
12. Add 100 µl of trisodium citrate and 100 µl of sodium sulphite to the stained chromosome preparation 15 min prior to analysis.

IV. COMMENTS
The procedure detailed in this article is directly applicable, with minor modification, to any actively dividing preparation of cells. Care should be taken to block the cells at a time during cell culture when the maximum number of cells are actively dividing and the time of incubation in colcemid should be adjusted to be approximately three-fourths of the time that the cells spend in the S phase of the cell cycle. Some cell types are resistant to disruption by vortexing (e.g., fibroblasts) and require gently syringing to release chromosomes into solution. The flow cytometer should be aligned according to the manufacturer's procedures such that the chromosomes first pass through the multiline ultraviolet beam required for Hoechst excitation and then, after the appropriate spatial separation, through the deep blue beam (457.9nm) required for excitation of chromomycin. A nozzle orifice diameter of 50-70 µm is recommended.

V. PITFALLS
Poor preparations that generate flow karyotypes without good separation of peaks are invariably produced when a low mitotic index is produced. Attention must be paid to culture conditions and the timing of subculturing and colcemid blocking to generate the highest mitotic index possible.

References
Gray, J. W., ed. (1989). "Flow Cytogenetics." Academic Press.

Sillar, R., and Young, B. D. (1981). A new method for the preparation of metaphase chromosomes for flow analysis. J. Histochem. Cytochem. 29, 74-78.

van den Engh, G., Trask, B., Cram, S., and Bartholdi, M. (1984). Preparation of chromosome suspensions for flow cytometry. Cytometry 5, 108-117.

van den Engh, G., Trask, B., Lansdorp, P., and Gray, J. (1988). Improved resolution of flow cytometric measurements of Hoechst- and chromomycin-A3-stained human chromosomes after addition of citrate and sulfite. Cytometry 9, 266-270.

van den Engh, G. J., Trask, B. J., Gray, J. W., Langlois, R. G., and Yu, L. C. (1985). Preparation and bivariate analysis of suspensions of human chromosomes. Cytometry 6, 92-100.