Bangs 2001

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  UNIT 4.1 Chromosome Preparation from CulturedPeripheral Blood Cells Chromosome preparations currently provide the only direct view of the genome as awhole. Although molecular methods allow a more detailed analysis of specific regions of the genome, the study of genetics is not complete without an appreciation of the metaphasecell. Peripheral blood provides a simple and reproducible source for large quantities of mitotic cells that may be used for clinical diagnosis (see UNITS 4.2 & 8.1 ) or studyingchromosome structure and organization (see UNIT 4.3 ).From a clinical point of view, the metaphase cell reveals not only numerical and structuralabnormalities, but also provides some biochemical information useful in diagnosinggenetic disorders. For instance, the DNA instability associated with Fanconi anemia ( UNIT 8.7  ) appears as increased chromosome breakage in metaphase cells; Bloom’s syndrome( UNIT 8.6  ) is manifested as increased sister chromatid exchange; and ataxia telangiectasiamay be spotted in metaphase chromosome preparations as infidelity in somatic recombi-nation. Chromosome preparations are essential for localizing genes and other DNAsequences on the physical map. This mapping is done directly by chromosomal in situhybridization or indirectly by correlating the presence or absence of a marker (such as anenzyme, a particular RNA, or a DNA sequence) with the presence or absence of achromosomal region.The stimulated T cell system described here is the most widely used means of obtaininglarge numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle inculture (basic protocol), combined with direct inhibition of chromosome condensation(alternate protocol), yield longer high-resolution prophase or prometaphase preparations.Such preparations are used for detailed analysis of microdeletions or subtle rearrange-ments, fine breakpoint analysis, and refined mapping. Although chromosomes may beobtained from other cells, human peripheral blood leukocytes are most amenable tosynchronization, and thus to high-resolution analysis of their chromosomes. Microscopeslide preparation of mitotic chromosomes from harvested cell culture suspensions is alsoexplained in the support protocol.  NOTE:  All reagents and equipment coming into contact with live cells must be sterile.  BASIC  PROTOCOL CULTURE AND METAPHASE HARVEST OF PERIPHERAL BLOOD T lymphocytes in whole blood are stimulated with the mitogenic plant lectin phytohe-magglutinin (PHA). The T lymphocytes “activate” to blast-like cells within 12 to 24 hrand continue to proliferate for 2 to 4 days. An optional cell-cycle synchronization stepcan be performed (see Fig. 4.1.1), which increases the number of cells harvested in earlymitosis. (This synchronization is not as involved as the one described in the alternateprotocol, which results in longer chromosomes and higher resolution). Metaphase cellsare obtained by treating cultures with Colcemid, a colchicine analog that disrupts thecentriole/spindle-fiber complex by interfering with microtubule formation. This treatmentresults in mitotic arrest, which in turn leads to an accumulation of cells in metaphase.Mitotic arrest is followed by treatment with a hypotonic KCl solution (hypotonic “shock”)to increase cellular volume. The cells are then fixed with methanol/acetic acid to removewater and disrupt cell membranes before being spread onto slides (support protocol). CPHG Contributed by Charles D. Bangs and Timothy A. Donlon Current Protocols in Human Genetics  (1994) 4.1.1-4.1.19Copyright © 2000 by John Wiley & Sons, Inc. 4.1.1 Cytogenetics   Materials For recipes, see  Reagents and Solutions  in this unit (or cross-referenced unit); for common stock solutions, see  APPENDIX 2 ; for suppliers, see SUPPLIERS APPENDIX  . Heparinized whole blood obtained via Vacutainer (Becton Dickinson)or syringe with preservative-free sodium heparin (25 U/ml)Complete RPMI/10% FBS medium (  APPENDIX 3G ) containing 50 µ g/ml gentamycinsulfate in place of penicillin and streptomycin100 ×  phytohemagglutinin-M (PHA) stock (GIBCO/BRL), reconstituted insterile deionized water (store at 4 ° C)10 µ M methotrexate (optional; see recipe)1 mM thymidine (optional; see recipe)10 µ g/ml Colcemid (GIBCO/BRL)75 mM KCl (0.56 g in 100 ml H 2 O; store ≤ 2 weeks at room temperature)Fixative: 3:1 (v/v) HPLC-grade absolute methanol/glacial acetic acid,(prepare fresh)15-ml sterile disposable conical polypropylene centrifuge tubesTB syringe equipped with 21-G needle (VWR Scientific)IEC HN-SII centrifuge with 958 rotor (or equivalent) CAUTION:  Human blood and methotrexate are hazardous; see  APPENDIX 2A  for guidelines on handling,storage, and disposal.  NOTE:  All incubations are performed in a humidified 37 ° C, 5% CO 2  incubator unless otherwise specified. Collect sample and initiate cultures 1. Collect peripheral blood by venipuncture into a sodium heparin Vacutainer or asyringe with 25 U preservative-free sodium heparin per milliliter of blood. Other anticoagulants, such as lithium heparin or EDTA are toxic to cells and should never be used. Samples should be shipped at room temperature. Blood in sodium heparin can beheld for ≤ 4 days and still be cultured successfully, but cultures are best initiated as soonas possible. If necessary, the specimen can be stored at 4 ° C. 2. Inoculate 0.25 ml of the whole blood obtained in step 1 (0.2 ml for newborns ≤ 3weeks old) into a sterile 15-ml centrifuge tube containing 5 ml complete RPMI/10%FBS medium, using a TB syringe equipped with a 21-G needle. Add 0.05 ml of reconstituted 100 ×  PHA solution.  Always replace the 25-G needle supplied with most TB syringes with a 21-G needle. Forcingblood through the narrower needle can lyse leukocytes. A single culture typically yields three to five full-slide preparations, or more if only part of the slide is used. Multiple cultures may be set up to meet clinical or research needs. 3. Incubate 2 to 4 days with tubes tilted at 45 ° . Three-day incubations are optimal, but 2- or 4-day cultures can be used to accommodatelaboratory scheduling concerns. Cultures from newborns will usually work well at 2 daysbut may also be harvested either directly or following a 1-day culture. Older patients’leukocytes require 3- or 4-day cultures because they do not seem to respond as quickly toPHA.Tilting culture tubes to 45 °  increases gas exchange and prevents cells from packing toodensely at the bottom. For these reasons it is best not to add more than 0.5 ml blood to anytube. Current Protocols in Human Genetics 4.1.2 ChromosomePreparationfrom CulturedPeripheralBlood Cells  Synchronize the cell cycle (optional) Synchronization (Fig. 4.1.1) is optional for routine blood culture and harvest, but willresult in longer chromosomes on average and more mitotic cells. The synchronizationpresented here (steps 4 and 5) will yield a greater number of mitotic cells; however, theseare not as long as those obtained using the high-resolution method (alternate protocol).4. On the day before harvest (e.g., after 4:00 p.m.), add 0.05 ml of 10 µ M methotrexate(10 − 7  M final) to block DNA replication. Incubate 16 to 18 hr (overnight).5. On the following day (e.g., at 8:30 a.m.), add 0.05 ml of 1 mM thymidine (10 − 5  Mfinal) to release the methotrexate block. Incubate ∼ 4 hr (e.g., until 12:30 p.m.). The exact hour of synchronization and release in steps 4 and 5 are not critical. However,because of methotrexate toxicity, the culture should be blocked no longer than 18 hr. Theexact interval between release of block and harvest is critical, because cells will progressquickly through G2, which lasts for 4 to 6 hr, to metaphase, which lasts ≤ 1 hr. mitosisprophaseinterphase: G1interphase: G2set up mixed cultures(asynchronous cell cycles);block with methotrexateperform thymidine releaseprepare slides fromsynchronous cell cycles S phase incubate16–18 hrmetaphase Figure 4.1.1 Cell culture synchronization. Two- or three-day asynchronous lymphocyte culturesare blocked by addition of methotrexate, a folate antagonist that prevents thymidine synthesis.Depletion of the thymidine pool prevents the cells from completing replication, and cells accumulatein the synthetic, or S, phase of the cell cycle. Subsequent addition of thymidine releases asynchronized wave of cells to complete replication and proceed through G2 and into mitosis. Current Protocols in Human Genetics 4.1.3 Cytogenetics   Harvest culture 6. Initiate harvest by adding 25 µ l of 10 µ g/ml Colcemid (0.05 µ g/ml final). Incubate30 min. For a 5-ml culture, this approximates two drops from a TB syringe equipped with a 25-Gneedle. If synchronization steps 4 and 5 have been omitted, the harvest can be initiated at any time3 to 4 days following the culture described in step 3. 7. Centrifuge 8 min at 180 ×   g  (1000 rpm in IEC 958 rotor), room temperature. Discardsupernatant.8. Add 6 ml of 75 mM KCl at room temperature and gently resuspend cells. Let stand15 min at room temperature. The amount of hypotonic solution to be added should be adjusted to the volume of the pellet.Some laboratories vary the length of hypotonic treatment. Increasing the time will increasechromosome spreading, but this treatment is a hypotonic “shock,” so that increasing theamount of hypotonic solution will have more impact than increasing the time of treatment. 9. Add 10 to 12 drops of fixative with a Pasteur pipet and mix well. Centrifuge as instep 7. This treatment serves to reduce the pH of the cells gradually to precondition them for the following fixation steps. It also lyses remaining red blood cells and begins the process of clearing resulting cellular debris. 10. Remove all but 0.5 ml of the supernatant and resuspend pellet in remaining super-natant by drawing it gently up and down with a Pasteur pipet. Add 1 ml fixative andimmediately mix gently. Adjust volume to 5 ml with fixative and mix thoroughly.Centrifuge as in step 7. The pellet after step 9 will be brown and clumpy because of erythrocyte debris. Resuspend gently but thoroughly to avoid clumped lymphocytes which may complicate slide-making. Do not draw too much volume into the pipet while resuspending because the cells will stick  permanently to glass. Do not press the pipet tip against the bottom of the tube when drawingand delivering the suspension, as this will lyse cells.The pellet after step 10 will be more homogeneous, and will usually have a light-brown towhite color. It may be ≤ 0.1 ml in volume. 11. Aspirate supernatant, resuspend pellet in 5 ml fixative, and centrifuge as in step 7.12. Remove supernatant and resuspend pellet in a volume of fixative sufficient to producea light milky suspension. Allow to stand 30 min at room temperature or storeovernight at 4 ° C.  Longer fixation will often improve chromosome spreading in difficult harvests. Keeping thesuspension overnight at 4 ° C can improve the quality of the preparation or can be done for scheduling reasons. Suspensions should be kept in polypropylene tubes containing plentyof fixative (e.g., 5 ml). Polystyrene tubes will react with fixative and should not be used. 13. Prepare slides and analyze chromosome spreads as described in the support protocol. Current Protocols in Human Genetics 4.1.4 ChromosomePreparationfrom CulturedPeripheralBlood Cells
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