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1. Introduction
The advent of chromosome banding techniques some 20 years ago (1,2) allowed the unequivocal identification of every chromosome in the human metaphase and provided a mapping scheme along each chro-mosome. Subsequently, a great deal of research has centered on preparing longer chromosomes with more bands visible. Chromosomes condense as they move through mitosis, and adjacent bands close up and appear to fuse. The earlier stages are longer with more bands recognized. It is not always possible to define the mitotic stage of a particular cell.
International standards have been agreed for various numbers of bands in the haploid set. Thus we have 400-,550-, and 850-band sets (3). Other workers report the use of even longer chromosomes (4,5). High-resolution banding has undoubted advantages in many fields. As well as allowing greater accuracy in traditional karyotype analysis, there are many reports of microdeletions and other abnormalities detected only on extended chromosomes (6). Likewise, in situ hybridization and gene localization techniques are taking advantage of the improved resolution.
The culture technique to prepare human chromosomes still follows the basic scheme laid down by Hungerford (7). Lymphocytes from peripheral blood are stimulated to divide in culture; cells are arrested in mitosis, swollen with hypotonic solution, fixed in an acid-alcohol fix, and spread on microscope slides by air-drying.
Published methods on the preparation of elongated chromosomes are abundant. As with banding techniques, different laboratories have preferences for particular methods and have developed their own variations. None of the methods is guaranteed to work with every specimen, being the nature of biological material. There is no doubt that it is very difficult for one laboratory to reproduce exactly all the conditions in another, and it is likely that published methods need some experimentation to optimize them for local conditions. Methods of preparation fall into three general categories. An individual protocol may use one or more of these approaches.
1.1. Induction of Synchrony
Stimulated peripheral blood lymphocytes in culture grow and divide asynchronously, and pass through the prometaphase and early metaphase stages relatively rapidly. Cells are blocked around S-phase (8) and, on release, will continue through mitosis in a wave of divisions, thus enhancing the potential yield of early stages. Methotrexate (9) and excess thymidine ( 1 0) are the most successful blocking agents. The timing of the interval between release of the block and harvest is the critical stage in the procedure and depends, in a complex way, on the various culture conditions. This is the main reason that many workers have found it difficult to duplicate successfully published methods or to maintain a high level of success with a particular scheme. To release the cells from the block, the blocking agent is removed, or at least overcome, and cells are encouraged to enter mitosis. The choice of release agent may be determined by the banding technique to be used subsequently.
1.2. Use of Chemicals to Affect the Condensation of the Chromosomes
Chromosomes progressively condense as the cell moves through mitosis. A number of chemicals have been found to counter this (12-14). Care must be taken to balance the reduction in contraction against
lowering of mitotic index and/or induction of chromosome aberrations.
1.3. Alteration of Arrest and/or Hypotonic
Treatments after Harvest (15-17)
Colcemid is used in most chromosome preparation techniques to destroy spindle formation and arrest cells in metaphase. Wiley et al. ( 17) questioned the necessity for co1cemid treatment, but most workers continue to use it in a variety of concentrations and exposure times.
Hypotonic treatment with 0.075M KCI still features in the majority of protocols, but many other formulations have been advocated, some with the specified aim of elongating chromosomes. There are various reasons why a particular method will be preferred for a line of research. However, in general, where time and material permit, it will be good practice to run tandem methods on each specimen. I will detail two protocols that have given good results.
2. Materials
2.1. Method 1
2.1.1. Supplemented Culture Medium
This will usually be prepared in bulk and aliquoted to culture vessels just before cultures are set up. The number of specimens to be processed will determine batch size. A week's supply is typical.
1. RPMI 1640 (GIbco, Gaithersburg, MD), 340 mL.
2. Fetal bovine serum, 60 mL.
3. Phytohemagglutlllm (HAI5 Wellcome, Dartford, UK), 4 mL.
4. PenicIllin and streptomycin (0.1 g and 100,000 U/mL) (Glaxo) mIxed solution, 0.4 mL. Store at 4°C.
2.1.2. Blocking Agent
Methotrexate injection (Lederle #4587-24) is obtained as 0.0025 g/mL (5 x 1O- 2 M) solution. A working solution (10- 5 M) is prepared by diluting 20 JlL with 9.980 mL sterile distilled water. This can be stored at 4°C
for several weeks. Methotrexate is a cytotoxic drug, and due care must be taken in handling.
2.1.3. Release Agents
1. ThymIdine (SIgma [St. Louis, MO] #T-9250) (l0-3M) is prepared by dissolving 2.42 mg/mL III stenle distilled water. Store at 4°C.
2. The alternative agent is 5-bromo-2'-deoxyundine Bdu (Sigma#B-5002):
Working solution (la- 2 M) IS prepared by dissolvmg 3 mg/mL m dIstilled water. Aliquots can be stored frozen for several months. Store vial III use at 4°C. Care must be taken in handlmg this teratogen and mutagen.
2.1.4. Arresting Agent
Co1cemid, 10 jlg/mL (Gibco). Store at 4°C.
2.1.5. Hypotonic Solution
KCI (0.075M) (1.4 g in 250 mL deionized water): Make up fresh for each harvest, and heat to 37°C.
2.1.6. Fix
Acetone-free methanol and glacial acetic acid are freshly mixed in the proportion 3: 1.
2.2. Method 2
2.2.1. Supplemented Culture Medium
This will usually be prepared in bulk and aliquoted to culture vessels just before cultures are set up. The number of specimens to be processed will determine batch size. A week's supply is typical.
1. RPMI 1640 (Gibco), 340 mL.
2. Fetal bovine serum, 60 mL.
3. Phytohemagglutmin (HAI5 Wellcome), 4 mL.
4. Penic1l1Ill and streptomycin (0.1 g and 100,000 U/mL) (Glaxo) mixed solutIOn, 0.4 mL Store at 4°C.
2.2.2. Inducing Agent
Actinomycin D (Sigma #A-1410) stock solution is prepared by dissolving 10 mg in I mL dimethylsulfoxide. Small aliquots are stored at -20°C. Working solution is 50 f...Ig/mL made by diluting thawed stock 200-fold in distilled water. This can be stored at 4°C for up to 2 wk. Actinomycin is poisonous, a known carcinogen and teratogen, and due care must be taken to avoid all contact.
2.2.3. Arresting Agent
Colcemid (Gibco) 10 jlg/mL. Store at 4°C.
2.2.4. Hypotonic Solution
KCI (0.075M) 1.4 g in 250 mL deionized water; Make up fresh for each harvest, and heat to 37°C.
2.2.5. Fix
Acetone-free methanol and glacial acetic acid are freshly mixed in the proportion 3: 1.
3. Methods
Aseptic laboratory procedures must be observed to avoid microbial contamination during the culture stages. All centrifugations are carried out in centrifuge with swing-out buckets in the rotor.
3.1. Method 1
1. Dispense 9.5 mL of supplemented RPMI 1640 medium (see Notes 1-4) Illto a stenle culture vessel (see Note 5), and inoculate with 0.75 mL of whole blood (see Note 6).
2. Incubate at 37°C for 72 h.
3. Inject the culture wIth 100 ilL methotrexate solutIOn (see Note 7), and remcubate at 37°C.
4. After 17 h, carefully remove the supernatant above the cell layer, preferably wIth a tube from a suctIon pump followmg the memscus. Pipeting can be used but IS more laborious.
5. Flick the flask wIth the fmger to dIstribute the cells, and resuspend in 9.5 mL supplemented medium.
6. Add 0.2 mL thymidine (see Note 8). Reincubate at 37°C for 3.75 h, or add 0.2 mL 5-bromo-2'-deoxyuridine (see Note 8). Reincubate at 37°C for 4.25 h.
7. MaIlltain the culture at 37°C while adding 60 ilL colcemid (see Note 9), and mcubate for a further 10 min.
8. Gently shake the flask, and transfer the culture to a comcal-based centrifuge tube.
9. Centrifuge at 150g for 10 min.
10. Suck off the supernatant to around 3 mm above the cell pellet.
11. Flick the tube to dIstnbute the cells and pipet m 10 mL of prewarmed KCI (see Note 10).
12. Incubate at 37°C for 10 min.
13. Centnfuge at 150g for 10 min.
14. Three layers should be visIble, a red cell pellet at the bottom, a slightly opaque layer of whIte cells, and the supernatant. Suck off the supernatant to within 3 mm of the whIte cell layer.
15. Flick the tube to loosen the cells. Use a vortex mIxer to stIr the cells more thoroughly while carefully addmg a pipetful of fIX dropwise to the middle of the vortex (see Note 11). Add two more pipetfuls of fix,
and allow to stand for at least 30 mm.
16. Centnfuge at 150g for 10 min, and remove most of the supernatant. Flick tube to resuspend cells, and add 2 pipetfuls of fIX.
17. Centnfuge at 150g for 10 min, and remove most of the supernatant.
Flick tube to resuspend cells, and add 1 pipetful of fix.
18. Centrifuge at 150g for 10 mIll, and remove supernatant to Just above cell pellet. Flick tube to resuspend cells, and add fix to give about 0.5 mL suspension.
19. Mix the suspension gently wIth a pipet and place a drop on a clean pohshed microscope shde (see Note 12). Allow to air-dry and examme under microscope to check cell density, spreading of chromosomes, and so forth. If cells are too densely packed, add more fix. If too sparse, spin down and reduce volume. Different methods of spreading may have to be adopted.
3.2. Method 2
1. Dispense 9.5 mL of supplemented RPMI 1640 medmm (see Notes 1-4) into a sterile culture vessel (see Note 5), and moculate wIth 0.75 mL of whole blood (see Note 6)
2. Incubate at 37°C for 68 h.
3. Inject the culture with 100 ilL actinomycm D solution (see Note 13), followed by 60 ilL colcemid solution (see Note 9), and reincubate at 37°C for 4 h.
4. Transfer to 15 mL conical-based centnfuge tube, and SpIll at 150g for 10 min.
5. Suck off the supernatant to around 3 mm above the cell pellet.
6. Flick the tube to distribute the cells, and pIpet in 10 mL of pre warmed KCl (see Note 10).
7. Incubate at 37°C for 10 min.
8. Centrifuge at 150g for 10 min.
9. Three layers should be visible, a red cell pellet at the bottom, a slightly opaque layer of white cells, and the supernatant. Suck off the supernatant to withm 3 mm of the whIte cell layer.
10. FlIck the tube to loosen the cells. Use a vortex mixer to stir the cells more thoroughly whIle carefully addmg a pipetful of fix dropwise (see Note 11). Add two more pipetfuis of fix and allow to stand for at least 30 min.
11. Centnfuge at 150g for 10 mm and remove most of the supernatant. FlIck tube to resuspend cells, and add 2 pipetfuls of fIX.
12. Centnfuge at 150g for 10 mm and remove most of the supernatant. FlIck tube to resuspend cells, and add 1 pipetful of fix.
13. Centnfuge at 150g for 10 min and remove supernatant to Just above the cell pellet. FlIck tube to resuspend cells, and add fix to give about 0.5 mL suspension.
14. Mix the suspension gently with a pipet and place a drop on a clean polished microscope slIde (see Note 12). Allow to air-dry, and examine under mIcroscope to check cell density, spreading of chromosomes, and so on. If cells are too dense, add more fix. If too sparse, spin down and reduce volume. DIfferent methods of spreading may have to be adopted.
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