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:

1. Grow cells to 85% confluence

2. Add colcemid to cell culture at 10 µl/mL

3. Incubate for 2 hours (depends on cell cycle: longer cycle = longer treatment; H9 = 30 min.)

4. Remove and collect growth medium and rinse cells with Hank’s or PBS

5. Add 2 mL trypsin and reincubate the cells at 37ºC, 5-7 min

6. Re-add the collected medium from step 4 to stop the trypsin and resuspend cells

7. Centrifuge the cells down at 1000 RMP, 6 min

8. Aspirate the medium but leave small amount of fluid

9. Flick the tube with finger to fully resuspend the cells

Addition of hypotonic solution:

1. Add 5 drops of pre-warmed hypotonic solution slowly against the side of the tube

2. Flick the tube with fingers until 1 mL had been added

3. Bring the volume to about 2 mL with hypotonic solution

4. Incubate at 37ºC, 7min

5. Centrifuge the cells down at 1000 RMP, 6 min

6. Aspirate the medium but leave small amount to resuspend the cells

Fixation:

1. Add 5 drops of fixative slowly against the side of the tube

2. Bring the volume to 2 mL with fixative, can vortex if see clumps

3. “Reverse bubble” to fully mix the cells

4. Fix the cells at RT, 30 min

5. Centrifuge, aspirate, and resuspend with finger as before

6. Remove the clumps (if any) by vacuum from the side of tube

7. Add fixative to 2 mL

8. “Reverse bubble” and let stand at RT, 20 min

9. Centrifuge, aspirate, and resuspend with finger as before

10. Add fixative to 2 mL

11. Cells are now ready to be dropped

Slide prepration (optional):

1. Rinse slide with ice-cold water

2. Rinse slide with fixative

3. Drop cells flat on slide (optimal humidity: 50%-60%)

4. Flood slide with fixative

5. 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.