The Mouse 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 or rat bone marrow, spleen, peripheral blood, or fetal liver. 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
• 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 (Table 1) 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.
   

  Table 1. Due to the different requirements for each product in the CFC assay, the recommended volume for each is listed.
  Reagent	Catalog #	Volume x 2*	Volume x 3**
  Methylcellulose Stock Solution	HSC001	1.4 mL	2.1 mL
  Mouse Methylcellulose Base Media	HSC006	2.7 mL	3.6 mL
  Mouse Methylcellulose Complete Media	HSC007	3.0 mL	4.0 mL
  *Volume for Duplicate Experiments **Volume for Triplicate Experiments

Procedure

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

Preparation of Mouse or Rat 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 or rat 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

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)	1 - 5 x 104	1 - 5 x 105
   Peripheral Blood	1 - 3 x 105	1 - 3 x 106
   Spleen	1 - 2 x 105	1 - 2 x 106
   Fetal Liver	1 - 5 x 104	1 - 5 x 105
   *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. With the exception of using the product HSC001, cell samples should be resuspended in IMDM/2% FBS. For the use of product HSC001, cell samples can be resuspended in the selected medium per instruction of each laboratory.
   2. 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 various methylcellulose-based media.

   Table 3. Volumes necessary for experiments using 35 mm culture dishes in duplicate or triplicate.
   	HSC001		HSC006		HSC007
   	Using cell samples in		Using cell samples in		Using cell samples in
   	Duplicates	Triplicates	Duplicates	Triplicates	Duplicates	Triplicates
   Methylcellulose-based Medium	1.4 mL	2.1 mL	2.7 mL	3.6 mL	3.0 mL	4.0 mL
   Culture Supplements or Cytokines Needed	1.6 mL	2.4 mL	0.3 mL	0.4 mL	None*	None*
   Cells Needed	0.3 mL	0.45 mL	0.3 mL	0.4 mL	0.3 mL	0.4 mL
   *Additional culture supplements or cytokines are not needed.

   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 8-12 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 Types

BFU-E (Burst forming unit-erythroid) Each colony is defined as clusters with a minimal of 30 cells that can be seen from day 7 onward. Each individual cluster consisted of tiny, irregular shaped cells that may appear fused together. Each cluster normally contains 5-8 cells, and the size of the cluster is similar to that of a single macrophage. The cluster may vary in sizes and color. A large BFU-E is usually bright red and is differentiable even without the use of a microscope. Smaller BFU-E may not appear red in color but is distinguishable based on the morphology.	BFU-E (40X) BFU-E (100X) BFU-E (200X)
CFU-GM (Colony forming unit-granulocyte, macrophage) The classification includes CFU-G (colony forming unit-granulocyte) CFU-M (colony forming unit-macrophage), and CFU-GM (colony forming unit-granulocyte, macrophage). Clonogenic progenitors of macrophages will give rise to a homogenous population of macrophages that are colorless, large, and round. Clonogenic progenitors of granulocytes will give rise to a homogenous population of granulocytes that are colorless and smaller than that of macrophages. The clonogenic progenitors or granulocyte, macrophage will give rise to a heterogenous population of macrophages and granulocytes. These developed colonies are colorless and consisted of round cells (granulocytes) and oval cells (macrophages). Each individual cell can be distinguished from erythrocytes by the size and color. The colony can be seen from day 7 onwards, and by day 12, the colony has drastically expanded in size.	CFU-M (left) and CFU-G (right) (40X) CFU-GM (40X) CFU-M (left) and BFU-E (right) (200X)
CFU-GEMM (Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte) Multi-lineage progenitors that give rise to the lineage of erythroid, granulocytes, macrophages, and megakaryocytes as the name indicated. It can be identified as reddish colored cells (erythroid) mixed with colorless cells (granulocytes, macrophages, and megakaryocytes) in a single colony. This progenitor is typically the largest colony on the culture dish; occasionally CFU-GM may attain a size comparable or larger than that of CFU-GEMM.	CFU-GEMM (40X)

Product Guide (For Use in Studies With Mouse and Rat Cells)