Karyotyping a Cell Line

Optimized by Noranne Enzer from Sorger Lab's Protocol, updated 2006

For more info on why each of these steps is critical, see this page.

This protocol works well for ES cells, primary cultures, or long-term cultures.

Prep work: Check that all solutions and supplies are in stock and slides are ready.

Prepare glass slides (5 per cell line) by soaking overnight (no longer) in EtOH:HCl 1:1.

Rinse with dH2O and soak in 200 proof EtOH until use.

Solutions

  • 10 mg/mL colchicin in EtOH, protect from light, store at -20c
  • 0.56% KCl
  • MeOH:Acetic Acid (3:1), stored at -20c
  • EtOH:HCL (1:1)
  • 200 Proof EtOH
  • Giemsa stain (Invitrogen cat#10092-013): 4% in Gurr Buffer
  • Gurr buffer-One tablet in 1 liter dH2O (Invitrogen cat# 10582-013)
  • Glycerol/PBS (1:1)

Supplies

  • coplin jar dedicated to Giemsa Staining
  • acid-washed glass slides
  • hotplate
  • pyrex dish (8x8 or 9x13 is fine)
  • 22mmx22mm coverslips
  • microscope with 40x or 63x objective, attached to computer to save images

Preparing cells

1-2 days before, plate cells onto one well of a 6-well plate or onto a 60mm dish. ES cells can be grown on feeders if needed. They will not interfere with the results.

1. Feed exponentially growing cells with media containing 10 ug/mL colchicin (this is a 1:1000 dilution of stock) and grow:

  • ES cells for 3-5 hours
  • Other cells for 8-12 hours (approximately ½ the time of a cell cycle)

2. Rinse with warm PBS, Trypsinize for 5 min. (make sure the cells are then in single-cell suspension by pipetting 10 times and checking under the scope. This is critical for getting good spreads) and Spin through 5mL media for 3 min at 800-1200 rpm (125g-280g).

3.Resuspend in 1ml PBS. Divide evenly between 2 eppendorf tubes. Spin for 2 min at 2K rpm (400g, lowest setting) in a benchtop microfuge.

4.Pull off PBS and resuspend each pellet in 500uL of 0.56% KCl. Sit at Room Temp 15min. Get ice for step 6

5.Spin for 2 min at 2K rpm (400g).

6.Remove KCl and resuspend pellets gently in cold MeOH:Acetic Acid 3:1 Let sit on ice for 10 min.

*Note: Try to pull the pellet into the pipet tip containing the MeOH to shear the pellet apart before it hits the MeOH.

7.Pellet 2 min at 3K rpm (850g), (*Note different conditions - the cells are now difficult to pellet, but if you spin too hard, the cells will break open in the tube)

8.Carefully aspirate pellet and resuspend in 200µL cold MeOH:Acetic Acid 3:1.(Can leave cells like this at 4°C overnight.)

Karyotyping

Dropping Cells

Prepare a steam bath by placing a pyrex dish (8x8 or 9x13) with 1-2 inches of dH2O onto a hotplate, adjusting the temp to get simmering and a lot of steam, but not boiling.

Dry prepared slides carefully with kimwipe before dropping cells.

Resuspend cells with gentle vortexing. Drop 10 ul of suspended cells with a pipetman from a height of approximately 8 inches onto a slide that is held at a 60 degree angle in steam. Tilt the slide to spread. Let the slide sit in steam while the MeOH evaporates off.

Let air dry. (Here you can quickly check under any 10x scope to see if it worked).

Place slides into coplin jar containing Giemsa stain for 30-40 min.

*Note: Giemsa stain is collected as hazardous waste.

Rinse in dH2O.

Take pictures at 40x or 63x. Save images on computer and print out each karyotype and count each chromosome by writing a number on each sister chromatid pair. This helps to ensure correct counting and provides a record for your notebook.

Giemsa stain: 4% in Gurr Buffer. Note- assume bottle from Gibco is 100%- Put 2 ml Giemsa in 48 mL Gurr, pour into coplin jar.

*Note: Giemsa stain is collected as hazardous waste.

Gurr buffer: One tablet in 1 liter dH2O

To preserve the slides for a long time, embed in 50% glycerol/50%PBS and secure coverslip.