Cytogenetics

V1 March 2012 V1 March M in HBSS: 4 vials M in HBSS: -5

M in distilled water: 4 vials containing 4 vials containing water: M in distilled -3 containing 1.5ml each containing 1.5ml each Methotrexate (Amethopterin), 10 (Amethopterin), Methotrexate Thymidine, 10 Warning! Methotrexate has caused adverse reproductive reproductive adverse has caused Methotrexate Warning! in humans. effects and foetal May irritation. tract skin, and respiratory eye, May cause genetic heritable abnormalities. May cause blood cause damage. not seen in routine analysis. High resolution allows more more allows High resolution analysis. not seen in routine of the karyotype. analysis detailed by the addition of methotrexate be synchronized can Cultures which blocks of thymidine biosynthesis (MTX), an inhibitor After cycle. of the cell in the S-phase (DNA synthesis) cells in are in the culture of the dividing cells most 16-18 hours, the MTX the culture, the S-phase. If thymidine is added to to synchronously proceed and the cells is released block short very may be added. A at which point colcemid mitosis, may with this technique conjuction in treatment colcemid chromosomes prometaphase extended produce be used to suspected. are or rearrangements when small deletions Materials 1. 2. and Stability Storage light. from and protected frozen be kept The solutions must 18 at least for stable the solutions are stored, If appropriately of preparation. the date months from Principle provide to method was developed karyotyping cell The blood abnormalities. Lymphocyte about chromosomal information divisions. In subsequent cell undergo not normally do cells to stimulated are lymphocytes of a mitogen, the presence a 48-72 hours, After by DNA replication. mitosis into enter in mitosis stop to the culture is added to inhibitor mitotic solution, by hypotonic treatment After stage. the metaphase can be microscopically chromosomes staining, fixation and abnormalities. for and evaluated observed is a special manipulation of the analysis High resolution provide designed to procedure karyotyping blood routine or prophase in late figures of mitotic number a large the chromosomes of mitosis stage At this prometaphase. G-banding, the After condensed. and less longer are of band resolution level greater show will chromosomes

Instructions for Use for Instructions

cytogenetic analysis cytogenetic For high-resolution high-resolution For

Kit Cell Synchronization Cell Synchronization

Store at: -20°C Store Cat. No.: 12-008-60 Procedure Related Products 1. Set up the blood culture according to the specific medium instructions (Cat. No.: 01-198-1 , 01-201-1). Product Cat. No. 2. Inoculate approximately 0.5ml of heparinized whole PB Karyotyping Medium 01-198-1 blood into a glass or plastic tube with 10ml of medium. Bone Marrow Karyotyping Medium 01-199-1 3. Incubate the culture for 72 hours. Hematopoietic Cell Karyotyping Medium 01-200-1 4. After 48 hours add (with carefull agitation), MTX Solution -7 -5 to a final concentration of 10 M (0.1ml from 10 M stock PB Karyotyping Medium with PHA-M 01-201-1 solution per tube). It is recommended to add the MTX in the afternoon - for overnight. Trypsin EDTA, 10X concentrate 03-051-5

5. After 17 hours, add (with continous vortexing) Thymidine Colcemid Solution 12-004-1 Solution to a final concentration of 10-5M (0.1ml from 10-3M stock solution per tube). 0.075M KCl Solution 12-005-1 6. After 5-5.5 hours, add 0.1ml of Colcemid Solution PHA-M 12-009-1 (Cat. No. 12-004-1) to each culture tube. If long, prometaphase chromosomes are desired, harvest after 10-20 minutes. If a high mitotic index is desired, harvest after 30-50 minutes of incubation. 7. Transfer the culture to a centrifuge tube and spin at 500g for 5 minutes. 8. Remove the supernatant and re-suspend the cells in 5-10ml of hypotonic 0.075M KCl (Cat. No. 12-005-1). Incubate at 37ºC for 10-12 minutes. 9. Spin at 500g for 5 minutes. 10. Remove the supernatant, agitate the cellular sediment and add drop-by-drop 5-10ml of fresh, ice-cold fixative made up of 1 part acetic acid to 3 parts methanol. Leave in 4ºC for 10 minutes. 11. Repeat steps 9 and 10. 12. Spin at 500g for 5 minutes. 13. Re-suspend the cell pellet in a small volume 0.5-1ml of fresh fixative, drop onto a clean slide and allow to air dry. 14. At this stage, the preparation can be stained with Orecin or Giemsa. Giemsa banding has become the most widely used technique. The most common method to obtain this staining is to treat slides with Trypsin-EDTA 10X (Cat. No. 03-051-5).

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