Protocols

Harvesting Cells:

*Hypotonic Solution:

  • 2 parts 0.6% Trisodium citrate
  • 1 part 0.075 M Potassium Chloride
  • make about 15ml per cell line
  • (pre-warmed to 37°C)

*Fixative Solution:

  • 3 parts methanol
  • 1 part acetic acid
  • make about 20ml per cell line
  • (ice cold)

* Make both solutions fresh daily

Be gentle when harvesting!!!

  1. If splitting cells every 2 - 3 days, add 10ul colcemid/ 10ml media for 30 to 35 hrs. 

    OR

    30ul colcemid/ 10ml media for ~19hrs. (usually gives best results)

    OR

    60ul colcemid/ 10ml media for ~8hrs.

    OR

    150ul colcemid/ 10ml media for ~3hrs. (not recommended unless short culture time is required)

    (Colcemid- Gibco, Cat. No. 15210-040, 10ug/mL)

  2. Place cell suspension into 50cc tube (or 15 cc tube for small cultures).
  3. Centrifuge at 800 rpm (137 x g), 6 min.
  4. Remove supernatant and add 5-7 drops of hypotonic solution that was prewarmed to 37°C

    Important to add the first few drops slowly to prevent lysing of the cells!

    • lightly tap to mix cells (should be no clumps)
    • add up to 5-7 ml more hypotonic solution while tapping to break up clumps
    • transfer to a 15cc tube (can combine pellets of same cell line here)
    • Incubate at 37°C for 10 minutes.
    • Add 5-7 drops of fixative solution (tube still contains the hypotonic solution) and invert tube to mix.  Do NOT vortex.
    • Centrifuge at 800 rpm (137 x g), 6 min.
    • Remove supernatant and loosen pellet by tapping tube. Add fixative to 15 mls, adding dropwise at first and constantly tapping to mix.  Fill the tube with fixative for shipping, normally we only add ~5ml fix.
    • Store at 4°C. Keep cool when shipping.