The Mouse Pre-B Colony Forming Cell (CFC) Assay Using Methylcellulose-based Media

The colony forming cell (CFC) assay, also referred to as the methylcellulose assay, is an in vitro assay used in the study of hematopoietic stem cells. The assay is based on the ability of hematopoietic progenitors to proliferate and differentiate into colonies in a semi-solid media in response to cytokine stimulation. The colonies formed can be enumerated and characterized according to their unique morphology.

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

• Cells derived from mouse bone marrow. Mice are routinely used between 6 - 12 weeks.
• Iscove’s Modified Dulbecco’s Media (IMDM)
• Fetal Bovine Serum
• IMDM/2% Fetal Bovine Serum
• (Optional) Flow Cytometry Mouse Lyse Buffer (R&D Systems, Catalog # FC003 or equivalent)

Materials

• 100 mm culture dishes
• 35 mm culture dishes
• 15 mL centrifuge tubes
• 10 mL syringes
• 3 mL syringes
• 5 mL vials (R&D Systems, Catalog # HSC999) 
• 16 gauge, 1½ inch needle
• 14 gauge laboratory pipetting needle (Popper & Sons, Catalog # 7941 or ThermoFisher Scientific, Catalog # 14-825-16M)
• Serological pipettes
• Pipettes and pipette tips

Equipment

• 37° C and 5% CO₂ humidified incubator
• Centrifuge
• Vortex mixer
• Hemocytometer
• Inverted Microscope

Reagent & Media Preparation

Note: Sterile technique is required when handling the reagents.

• Methylcellulose-based Media - Thaw the bottle of media at 2 - 8° C overnight. After the media is completely thawed, shake the bottle vigorously to thoroughly mix the contents. Allow air bubbles to escape by placing the bottle either at room temperature or at 2 - 8° C for 0.5 - 1 hour.
  
  Aliquot the exact amount of media required for a single experiment into sterile 5 mL vials using a sterile 14 gauge laboratory pipetting needle and a 10 mL syringe.
  
  Note: Due to the high viscosity of methylcellulose media, the use of a syringe is necessary to accurately measure volume. The 14 gauge laboratory pipetting needle referred to in the Supplies Required section is recommended due to its larger diameter. The needle is autoclavable and reusable.
  
  Store the aliquots at -20° C in a manual defrost freezer until use. Do not use past the kit expiration date.
   

Procedure

Use sterile technique. Use serological pipettes to transfer and remove solutions.

Preparation of Mouse Bone Marrow Cells

Note: When handling biohazardous materials such as sharp needles, safe laboratory procedures should be followed and protective clothing should be worn.

1. Prepare a suspension of mononuclear cells from mouse bone marrow using traditional methods. Both femurs and tibiae from one mouse typically yield 2.0 - 6.0 x 10⁷ hematopoietic cells. A detailed protocol can be found in Current Protocols in Immunology, Isolation of Murine Macrophages (1994) Coligan, J.E. et al. eds. John Wiley & Sons, Inc., Volume 3, Supplement 11, 14.1.4.
2. To remove cell clumps and debris after harvesting the bone marrow cells, pass the cell suspension through a 70 μm nylon strainer.
3. After filtration, the cells should be used as soon as possible. Or, if desired, the red blood cells (RBC) can be removed. To lyse RBC, use Flow Cytometry Mouse Lyse Buffer (R&D Systems, Catalog # FC003) according to the instructions.
4. Wash the cells in 50 mL centrifuge tubes with room temperature IMDM/2% FBS by centrifuging at 300 x g for 8 minutes. Remove the supernate completely and resuspend the cells in 10 mL of IMDM/2% FBS by gentle pipetting, to generate a single cell suspension.

Note: Bone marrow cells should be used as soon as possible or frozen according to the standard freezing protocol used in each laboratory.

Methylcellulose Assay

Figure 1

1. Thaw aliquots of methylcellulose-based medium at room temperature for approximately 30 minutes. Allow the vials to thaw without disturbance.
2. During the thaw step, resuspend the cell sample in 10 mL of IMDM/2% FBS or in an appropriate volume and count.
3. Calculate the total number of cells needed in the experiment using Table 2 to determine the recommended final cell number per 35 mm culture dish. Transfer the appropriate volume of cells (plus a slight excess) into a new 15 mL conical tube. Centrifuge for 8 minutes and 300 x g.

   Table 2. Determining the approximate cell number needed for each 35 mm culture dish.
   Sample Source	Final Cell Number*	Stock Cell Number (10x Final)
   Bone Marrow (untreated)	5 x 104 - 1 x 105	5 x 105 - 1 x 106
   *Final cell number per 35 mm culture dish (or 1.1 mL of media)
   Note: The cell plating numbers listed above serve as a reference only. Optimal cell plating concentration should be determined by each laboratory for each cell type.

4. Remove the supernatant and resuspend the cells in IMDM/2% FBS or the appropriate medium to the desired cell concentration for plating (usually 10X the recommended cell number required per 35 mm culture dish listed in the Cell Plating Number Chart).
   1. The optimal cell plating concentration should be determined in the initial experiment by including a lower and higher cell concentration than the cell concentration recommended in the Cell Plating Number Chart.
5. The table below provides the recommended volumes of cells from the 10x stock and additional culture supplements or cytokines to be added to the methylcellulose aliquots. The final concentration of methylcellulose before adding cells should be approximately 1.3% for the methylcellulose-based medium.
    

   Table 3. Volumes necessary for experiments using 35 mm culture dishes in duplicate or triplicate.
   	Using cell samples in
   	Duplicates	Triplicates
   Methylcellulose-based Medium	3.0 mL	4.0 mL
   Cells Needed	0.3 mL	0.4 mL

    

   Figure 2

6. Vigorously vortex the vial to thoroughly mix the cells with the media.
7. Wait for approximately 20 minutes before continuing with the procedure to allow air bubbles to escape.
8. Add 1.1 mL of the final cell mixture into 35 mm culture dish using a 3 mL syringe fitted with a 16 gauge needle. Spread the media evenly by gently rotating the dish.
   Make sure to use non-tissue culture treated petri dishes.
9. Place two sample dishes and an uncovered dish containing 3 - 4 mL of sterile water in a 100 mm culture dish and cover. The sterile water dish serves to maintain the humidity necessary for colony development.
10. Incubate the cells for 6 - 8 days at 37° C and 5% CO₂. Avoid disturbing the dish during the incubation period to prevent shifting of the colonies.

Figure 3

Colony Scoring

Score colonies at the end of the incubation period. Identify and count individual colonies using an inverted microscope and a scoring grid.

• Prepare the scoring grid as described in the Scoring Grid section. The diagram provided below can be used as a template to reproduce the scoring grid on a 100 mm culture dish. Mark the grid on a new 100 mm culture dish by placing the culture dish on the template and tracing the grid with a marker or pen.
• Refer to the Counting Criteria section for guidance to identify and count colonies.

Scoring Grid

Figure 4

Counting Criteria

Colonies consisting of at least 30 cells are counted (or the minimum cell count set by each laboratory).

Colony Type

Product Guide (For Use in Studies With Mouse Cells)