METAPHASE SPREAD: 11/2/04 Materials: Colcemid (GIBCO: cat #: 15210-040) Hypotonic solution (pre-warmed 37C): 1:1    0.4% KCl + 0.4% Sodium citrate Fixative: 3:1  MeOH + Acetic acid (e.g., 15 mL MeOH + 5 mL Acetic acid) Pasteur transpipet Procedure: Grow and harvest cells: - Grow cells to 85% confluence
- Add colcemid to cell culture at 10 µl/mL
- Incubate for 2 hours (depends on cell cycle: longer cycle = longer treatment; H9 = 30 min.)
- Remove and collect growth medium and rinse cells with Hank’s or PBS
- Add 2 mL trypsin and reincubate the cells at 37ºC, 5-7 min
- Re-add the collected medium from step 4 to stop the trypsin and resuspend cells
- Centrifuge the cells down at 1000 RMP, 6 min
- Aspirate the medium but leave small amount of fluid
- Flick the tube with finger to fully resuspend the cells
Addition of hypotonic solution: - Add 5 drops of pre-warmed hypotonic solution slowly against the side of the tube
- Flick the tube with fingers until 1 mL had been added
- Bring the volume to about 2 mL with hypotonic solution
- Incubate at 37ºC, 7min
- Centrifuge the cells down at 1000 RMP, 6 min
- Aspirate the medium but leave small amount to resuspend the cells
Fixation: - Add 5 drops of fixative slowly against the side of the tube
- Bring the volume to 2 mL with fixative, can vortex if see clumps
- “Reverse bubble†to fully mix the cells
- Fix the cells at RT, 30 min
- Centrifuge, aspirate, and resuspend with finger as before
- Remove the clumps (if any) by vacuum from the side of tube
- Add fixative to 2 mL
- “Reverse bubble†and let stand at RT, 20 min
- Centrifuge, aspirate, and resuspend with finger as before
- Add fixative to 2 mL
- Cells are now ready to be dropped
Slide prepration (optional): - Rinse slide with ice-cold water
- Rinse slide with fixative
- Drop cells flat on slide (optimal humidity: 50%-60%)
- Flood slide with fixative
- Dry slide on wet paper towels
Drop the cells onto pre-cleaned slide or treated slide above, rinse with fixative (optional), dry on wet tower papers, and observe for metaphase spreads with a phase-constrast microscope. |