CAPITULO III SISTEMA DE COORDINACIÓN FISCAL
3.4 OTROS FONDOS
HARVESTING OF CHROMOSOMES2
Cells are subjected to hypotonic treatment, which increases their volume, and disrupts the cell membrane of the red blood cells allowing their removal.
A fixative solution is added to the cell suspension to preserve the cells in their “swollen” state and to remove the water, thus “hardening” the biologic material. The common fixative (3:1 methanol:acetic acid) removes lipids and alters/denatures proteins thus making the cell membrane remnant very fragile, which is important for subsequent chromosome spreading.
Metaphase Chromosomes
The mitotic spindle formation is blocked, usually by adding colcemid to the culture, and the cell division is stopped at the metaphase level.
Prometaphase Chromosomes
Methotrexate is used to block the cells in the late S phase to get elongated chromosomes with a resolution of 850 bands when compared to the metaphase chromosomes. The S phase block is released by thymidine synchronization.
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Requirements
1. Methotrexate (MTX) solution
MTX stock solution (1 × 10–3 M)
MTX [(+) Amethopterin] 1 mg
Distilled water (Sterile) 2.2 mL Store in frozen condition in dark vials
MTX working solution (1 × 10–5 M)
MTX stock solution (1 x 10–3 M) 0.1 mL Distilled water (Sterile) 9.9 mL
MTX is relatively unstable and can be used for one month. Store frozen in dark vials. Sterilization is usually not required.
2. Thymidine (1 × 10-3 M)
Thymidine 2.5 mg
Distilled water (Sterile) 10 mL
Filter sterilize and store frozen in small aliquots. Solution is stable and can be used for one month. Both the solutions have to be left in dark for thawing before use.
3. 0.02% colchicines - 1 mg in 5 mL of sterile water (available commercially). Store frozen in amber colored bottles (4°C )
4. 0.56% Potassium chloride solution (0.075 M) - Prepared freshly and prewarmed (37°C) before use.
5. Carnoy’s fixative - 1:3, acetic acid: methanol. For one tube approximately 20 mL of fixative is required for one tube. Based on the number of tubes fixative is prepared freshly before use and left in the freezer till use.
6. Centrifuge tubes 3 Nos 7. Pastuer pipettes 3 Nos 8. Micropipettes (5–50 μL) 1 No
9. Timer 1 No
10. Cold slides 4 slides per tube (approx.)
11. Forceps 1 No.
12. Diamond marker 1 No.
13. Hot plate 14. Centrifuge
15. Water bath set at 37°C 16. CO2 incubator.
Procedure
1. After 72 hours of incubation, add 0.1 mL of MTX working solution (1x 10-5 M) to the culture. (Final concentration of MTX is 1x10 - 7 M.) 2. Incubate the cultures at 37° C for 17 hours (overnight)
3. Centrifuge the cultures at 1,800 rpm for 8 minutes and discard the medium containing MTX.
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4. Wash the cells twice by suspending them in fresh unsupplemented medium (medium without serum), by centrifuging (1,800 rpm for 10 minutes) and then discarding the supernatant each time. After the final wash, suspend the cells in complete culture medium containing the serum and antibiotics at usual concentrations.
5. Transfer the contents to a culture vial and add 0.1 mL of thymidine solution to each culture containing 10 mL of medium.
6. Sterilize the mouth of the culture vials and incubate the cultures at 37°C for 5 hours in the carbondioxide incubator.
7. After the completion of 5th hour, take the culture vial and leave it in vial stand in the laminar airflow hood.
8. Sterilize the mouth of the vials and allow them to cool.
9. Add 100 μL of colchicine to culture containing 10 mL of medium, gently mix and incubate at 37oC in the carbon dioxide (CO2 ) incubator for 10 minutes.
10. Take the tubes outside the incubator and leave them in the laminar airflow hood.
11. Flame the mouth of the vials and allow them to cool.
12. Take the sterile centrifuge tubes and label them with the name of the patient.
13. Flame the mouth of the centrifuge tubes and allow it to cool.
14. Then transfer the contents from the vial to the centrifuge tubes.
15. Centrifuge the tubes at 1,800 rpm for 10 minutes.
16. Discard the supernatant by pipetting leaving as little medium as possible over the cell button.
17. Resuspend the cell button in 5 mL of prewarmed hypotonic solution.
Close the mouth of the centrifuge tubes with aluminium foil and incubate for 30 minutes in a water bath at 37°C.
18. Add 4–5 mL freshly made cold fixative to each tube and mix gently.
Centrifuge at 1,800 rpm for 10 minutes. [Cells should be handled very gently following hypotonic treatment. Any harsh treatment may rupture the cells, leading to many incomplete metaphases in final preparations. To prevent any such undue damage to the cells, avoid passing the suspension through narrow-tipped droppers or pipettes at this stage].
19. Discard the supernatant. Disturb the pellet thoroughly by tapping at the bottom of the tube. Resuspend the pellet in 5 mL of fresh cold fixative.
20. Again centrifuge the tubes, discard the supernatant, and suspend the cells in fresh cold fixative. Repeat this twice.
21. After the final centrifugation, suspend the cells in a small volume of fixative (0.5–1.0 mL) to make a suspension.
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22. Place 3–4 drops evenly on a cold wet slide and allow it to dry. Place the slide on a slide warmer immediately after the cell suspension is dropped on the slide (50–60°C). After the slide is completely dry, examine under low magnification (10 X) to check the cell density and spread of metaphase chromosomes. If the cell density is too high, add a few more drops of fixative to the cell suspension. If the cell density is low, centrifuge the suspension and re-suspend the pellet in a smaller amount of fixative. The slides for use are described in Process No. 1) 23. Then the slides are marked at one end (usually at right hand corner) with the following details: G-No (Genetic reference No.), Date, method of culture with the slide numbers (96 hr represented by “HR” meaning
“High Resolution”; slide Nos given according to the order of the slides prepared (HR-1, HR-2, etc.).
24. After all the slides are checked, dried and kept inside the slide box.
25. Allow the slides to age for at least one week or incubate the slides at 60oC for sixteen to seventeen hours to do the staining and banding.