Harvesting cells from CFU assays

It is possible to perform additional analysis on cells that have been cultured in colony-forming unit (CFU) assays. Cells from colonies grown in methylcellulose-based medium can be harvested for further analysis or culture in two ways: plucking or washing them out of the culture medium.

If the cells of a particular colony are desired for downstream analysis, pluck individual colonies as follows:

  1. Identify and locate the colonies of interest under the microscope.
  2. Pluck each colony using a P200 micropipettor (set to a limited volume, i.e. 50 μL, to ensure only the desired colony is plucked). Be sure not to release the pipette in the methylcellulose-based medium after collecting the colony into the pipette tip.
  3. Place the plucked colonies into microcentrifuge tubes containing 400-500 μL of liquid medium (i.e. Iscove's MDM or PBS), or into individual wells of a 96-well plate (preferably U or V-shaped wells) containing 100-200 μL of liquid medium.
  4. Gently pipette up and down to wash the cells from the pipette tip.
  5. Wash the cells twice in an excess of liquid medium to ensure that the methylcellulose is washed away.
  6. Resuspend the cells to the appropriate concentration for your application.


If all of the cells in the culture are required for subsequent experiments, harvest all the colonies in the dish as follows:

  1. Move the culture dishes to 4°C for a couple of hours.
  2. Add 1 mL of liquid medium (i.e. Iscove's MDM or PBS) to each dish.
  3. Pipette up and down gently to mix the methylcellulose-based medium with the liquid medium (using a P1000 micropipettor).
  4. Dispense the diluted methylcellulose-based medium and cells into a centrifuge tube (i.e. 15 mL conical tube).
  5. Add another 1 mL of liquid medium to the dish to wash away any remaining cells and add this to the tube.
  6. Top up the tube with an additional 5 mL of liquid medium to ensure that the methylcellulose is completely diluted.
  7. Centrifuge at 350 x g (~1200 rpm) for 10 minutes to pellet the cells.
  8. Resuspend and wash the cells again to remove any remaining methylcellulose.
  9. Resuspend the cells to the appropriate concentration for your application.


Please refer to our website for more information on MethoCult methylcellulose-based media products optimized for human, mouse and rat hematopoietic CFU assays.

For further assistance and information, please contact techsupport@stemcell.com.