MITOTIC CHROMOSOME PREPARATIONS FROM MAMMALIAN SUSPENSION CELL CULTURES

 

EQUIPMENTS
  • Acid-cleaned slides from protocol 4

 

REAGENTS
  1. Colcemid: stock solution: 0.01% (w/v) in sterile distilled water
  2. Hypotonic solution: 0.075 M potassium chloride, or 0.8-1.2% citrate solution (e.g. tri-sodium citrate) or 1 part culture medium (as used for growing the cells) + 3 parts distilled water
  3. Alcohol:acetic acid fixative: 3 parts 96% ethanol (or 100% methanol) to 1 part glacial acetic acid (prepare immediately before use; do not store for more than 30 min).

 

MATERIAL
Short or long term cell culture

 

METHODS

Sterile conditions are recommended until cells are fixed (Step 5).
  1. Add colcemid stock solution to 10-40 ml culture (1-10x10 6 cells) to give final concentration of 0.01%. Incubate for 1-2 h at 37°C or cell growth temperature to accumulate metaphases (Note 2).
  2. Transfer cells into one to three 15 ml sterile glass or polypropylene centrifuge tube and centrifuge for 10 min at 1000g (Note 3).
  3. Carefully pour off the supernatant fluid. Resuspend the cell pellet with the last drop of supernatant by shaking. Do not use a pipette. Slowly add about 10 ml pre-warmed (37°C or room temperature) hypotonic solution and leave to stand for 10-20 min at 37°C or 20-40 min at room temperature (Note 4)
  4. Centrifuge at 1000 g for 10 min. Pour off supernatant and shake up pellet
  5. Resuspend in about 10 ml of fixative. Add the first 1 ml of fixative dropwise and shake well after each drop. Leave for 10 min at room temperature (Note 5).
  6. Repeat steps 4 and 5 two to five times (Note 6).
  7. Cells can be stored in the refrigerator at this point for several days
  8. For spreading, centrifuge the suspension (step 4) and resuspend in 0.5-1 ml of fixative. Transfer or drop from a height of a few centimeters, one or two drops of well­ mixed suspension to a clean slide . Gently shake the slide to distribute cells and blow on them gently. Let dry in an upright position
  9. View under a phase contrast microscope to check cell density and quality of spread (Note 9). Spread rest of the suspension on slides .
  10. Screen slides under a phase contrast microscope (Protocol 5.8)
  11. Store slides desiccated at 4°C (or -20°C) if necessary
NOTES
  1. Subculture long-term cell cultures one day before preparation of chromosomes, so that the maximum growth rate is reached on the day of preparation. For short-term blood cultures, blood is collected in heparin tubes and lymphocytes stimulated with phytohemagglutinin. Diagnostic labs will certainly have procedures set up that use most commonly 5 ml of blood, separate lymphocytes and count cells for accurate stimulation and growth. Kits are available (e.g. from Difco) using a few drops of blood to yield plenty of slides .
  2. Time and concentration needs to be adjusted for different cell types .
  3. The pellet should be about 20-50Ál. Too small a pellet can be lost during repeated centrifugation. Some delicate cells will need centrifugation at lower speeds
  4. Hypotonic treatment swells the cells and facilitates untangling of the chromosomes and hence spreading. Time and temperature are critical and vary for cell type and species. Extended treatment and higher temperatures increase swelling of cells. Under-treated cells do not spread well; over-treated cells burst too early and chromosomes are lost
  5. If fixative is added too quickly cells clump and normally can not be separated again
  6. Centrifugation of the suspension in fixative should be slightly harder than before. Be careful that you do not shake up the pellet before supernatant is poured off. Fixing time can be reduced after the 2nd round, but make sure that overall time in fixative is at least 30 min
  7. When the fixed cell suspension is dropped onto the surface of the glass slide, the fixative disperses and evaporates quickly, and the forces generated spread the cells, nuclei and chromosomes. This step can be monitored under phase contrast and adjusted to give well-separated chromosomes in complete metaphase plates. Shaking and blowing the slide increases the spreading forces, while gentle dropping and handling keep the chromosomes together. The density of the cell suspension is also important for effective spreading
  8. Some researchers store slides in ice cold water and drop cells on the wet slides
  9. If chromosomes can not be separated and spread by increased blowing and shaking, are too dense or sparse, or chromosomes are surrounded by a lot of cytoplasm or other 'dirt', add 10 ml of fixative and repeat step 4 and 5
  10. Hypotonic treatment, now used almost universally for swelling cells and disentangling chromosomes was described by Hsu (1952), and was, according to his postscript, an accidental finding. The protocol here is modified from Schwarzacher (1974).