Colony-forming unit (CFU) assays can be used to examine the proliferation and differentiation ability of hematopoietic stem and progenitor cells. It is also possible to perform additional analysis on cells that have been cultured in CFU assays. Cells from colonies grown in methylcellulose-based medium (e.g. MethoCult™ H4034 Optimum, Catalog #04034) can be harvested for further analysis or culture in two ways: plucking or washing them out of the culture medium. If cells of a particular colony are desired for downstream analysis, individual colonies can be plucked from CFU assays. Alternatively, if all cells in the culture are required for subsequent experiments, all colonies may be harvested from the dish. This protocol describes both of these procedures for harvesting cells from CFU assays for downstream analysis.

Option 1: Plucking Individual Colonies for Downstream Analysis

Materials

• Phosphate-buffered saline (PBS; or Iscove's Modified Dulbecco's Medium, Catalog #36150)
  • Note: A serum-free liquid medium (e.g. IMDM with 25 mM HEPES, Catalog #36150) may be used if the CFU assay was performed using a serum-free MethoCult™ formulation
• 0.5 mL microcentrifuge tubes (e.g. Costar® Microcentrifuge Tubes, Catalog #38088) or 96-well plate (preferably U- or V-shaped wells, e.g. Corning® 96-Well Round-Bottom Microplate, Catalog #38018)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)

Protocol

1. Add 400 μL PBS (or IMDM) to 0.5 mL microcentrifugetubes or 100 - 200 μL PBS or IMDM to individual wells of a 96-well plate.
2. Place the culture dish with colonies of interest on the stage of the inverted microscope. Using the 4X objective, locate the colony to be harvested. With a 20 µL or 200 µL pipettor and a pipette tip (non-sterile tip is acceptable) pick ~20 - 50 μL of the colony of interest. Be sure not to release the pipette in the methylcellulose, or bubbles will form. Dispense the colony into the tube containing PBS or IMDM. Pipette up and down several times to ensure that cells from the colony do not remain in the tip of the pipette.
   
   
   Note: Do not let cells sit in PBS at room temperature for longer than 1 hour.
3. Centrifuge at ~350 x g for 10 minutes.
   
   
   Note: If using microcentrifuge tubes, place the tubes in the same direction with the front of the cap facing inward, so that the colony will pellet at the back of the tube where contact is more easily avoided when removing supernatant.
4. Remove and discard supernatant. Resuspend the pelleted colony with IMDM or PBS, using 500 μL/microcentrifuge tube or 100 - 200 μL/well.
5. Perform the wash (step 3) one more time. Remove and discard supernatant.
6. Resuspend cells to the appropriate concentration for downstream applications.

Option 2: Harvesting All Colonies for Subsequent Experiments

Materials

• Iscove's MDM with 2% FBS (IMDM + 2% FBS, Catalog # 07700) or PBS or other suitable liquid medium
  • Note: A serum-free liquid medium (e.g. IMDM with 25 mM HEPES, Catalog #36150) may be used if the CFU assay was performed using a serum-free MethoCult™ formulation
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
• Centrifuge tube (e.g. Falcon® Conical Tubes, 15 mL, Catalog #38009)

Protocol

1. Using a 1 mL pipettor, add 1 mL of IMDM + 2% FBS (or PBS, or other suitable liquid medium) to each 35 mm culture dish.
2. Pipette up and down gently to mix the methylcellulose-based medium with the cells (using a 1 mL pipettor or a 1 mL serological pipette).
3. Transfer the diluted methylcellulose and the cells into a centrifuge tube (e.g. 15 mL conical tube).
4. Add an additional 1 mL of IMDM + 2% FBS or liquid medium of choice to the 35 mm dish. Mix thoroughly by pipetting up and down to collect any remaining cells. Collect this fraction and add to the tube from step 4.
5. Top up the tube to 10 mL with IMDM + 2% FBS to ensure that the methylcellulose is completely diluted.
6. Centrifuge at ~350 x g for 10 minutes. After centrifuging, you should observe a pellet at the bottom of the tube.
7. Remove the supernatant. Cells are ready for use in downstream applications, or repeat steps 6 - 7 to remove any remaining methylcellulose.
8. Resuspend cells to the appropriate concentration.