Method: Harvesting Cultured Lymphocytes for In-Situ Hybridization Metaphases

Rosalie Veile

Principle:

There are 3 steps to the harvesting procedure:
1. Dividing cells are arrested in mitosis with the use of colcemid. Colcemid prevents the formation of the spindle apparatus responsible for cell division, thereby permitting an accumulation of metaphases. Note: Lymphoblastoid cell lines can also be prepared for chromosome analysis, because the morphology of lymphoblastoid cells is comparable to lymphocytes prepared from whole blood samples.
2. The addition of the hypotonic solution, potassium chloride (KCl), will result in water rushing into the cells due to the concentration gradient caused by the less-than-physiological concentration outside of the cells. The cells will swell and cytoplasmic membranes will stretch. The cells resemble a water balloon with chromosomes suspended inside.
3. With the addition of fixative (1:3:: methanol:acetic acid), each cell is preserved in its swollen shape. Chemical changes make the cell harden (not as fragile as in hypotonic), but remaining a swollen sac of suspended chromosomes.

Time required:

2-3 hours when harvesting 8-12 culture tubes on day 4 of chromosome preparation (on days 1-3, the lymphocytes are stimulated to divide in culture media).

Special Reagents:

• Colcemid (Gibco, Cat. No. 120-5211 AD)

Procedure:

1. On day 4, or 72 hours after the culture was initiated (see "Culturing lymphocytes for metaphases used in in-situ hybridization", day 1-3), prewarm 0.075 M KCl at 37 degrees C and chill a 500 ml bottle of methanol to 4 degrees C.
2. After 72 hours of culture incubation, add 0.25 ml colcemid (10 µg/ml) with a 1cc syringe to each 15 ml tube. Invert tubes several times to mix. Return cultures to the 37 degrees C incubator for 10-20 minutes. Note: The length of chromosomes is dependent on the concentration of colcemid and the time of mitotic arrest. The higher the concentration of colcemid and the longer the time of mitotic arrest, the shorter the chromosomes will be. Therefore, it is best to keep the concentration of colcemid and the time of exposure to a minimum. Colcemid will continue to be active until the addition of fixative. Phytohemagglutinin (PHA) stimulated cultures require a very short time of exposure (10-30 minutes) to colcemid because they are rapidly-dividing cells. The length of chromosomes should be greater than at the 500 band stage (elongated).
3. Resuspend the cells again by inverting the tubes and centrifuge cultures for 8 minutes at 1200 rpm in the Beckman TJ-6 centrifuge.
4. Aspirate the supernatant with a sterile pasteur pipet connected to a vacuum apparatus. Resuspend the cells with a bulbed 9 inch pasteur pipet. Slowly add 1-3 ml of warm hypotonic solution. Continue to add 1-3 ml at a time, until 10 ml of KCl has been added. Blow air into the suspension with a bulbed pipet after each addition of KCl solution. Incubate the tubes for 18 minutes at 37 degrees C.
5. Add 1 ml of freshly prepared cold fixative (1:3 :: methanol, 4 degrees C: acetic acid) to each culture very slowly (drop by drop). Bubble air through the suspension after every few drops. This will quickly fix the cells in a swollen shape and begin lysing red blood cells.
6. Centrifuge the cells for 8 minutes at 1200 rpm in the TJ-6 centrifuge.
7. Aspirate the supernatant (contains lysed red blood cells and cellular debris). Resuspend the cell suspension by bubbling air through the pellet and add 1-3 ml of fixative. Continue washing cells with fixative, until a total of 10 ml of fixative has been added.
8. Repeat step 6 and step 7, three more times or until all red cells are lysed and a white cell pellet is observed.
9. Prepare microslides for in-situ hybridiation by dropping the harvested metaphases to the absolute ethanol rinsed microslides (refer to microslide preparation procedure). For best results, this step should be done immediately after the metaphases are harvested. If microslides cannot be prepared immediately, the cell suspensions can be stored in the fixative solution at 4°C for 24 hours.

Solutions:

• Colcemid solution:
  Colcemid is prepared in PBS and lyophilized (10 µg/ml). Rehydrate with 10 ml sterile, deionized, distilled water (ddH2O). Store at 4 degrees C for up to 6 months.
• 0.075 M Potassium Chloride (KCl):
  Dissolve 5.6 g KCl in 1000 ml sterile ddH20. Store at 4 dergees C for up to 1 month. Prewarm to 37 degrees C several hours prior to use.
• Fixative:
  Prepare fixative by mixing 75 ml of 4 degrees C methanol with 25 ml glacial acetic acid. Fixative should be made fresh before each use, because it can absorb water upon standing and the pH changes with time.

References:

Monteleone, P., Department of Genetics, Cardinal Glennon Children's Hospital.