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.
Note: Sterile technique is required when handling the reagents.
|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|
|Human Methylcellulose Base Media||HSC002||2.7 mL||3.6 mL|
|Human Methylcellulose Serum-Free Base Media||HSC002SF||2.7 mL||3.6 mL|
|Human Methylcellulose Complete Media||HSC003||3.0 mL||4.0 mL|
|Human Methylcellulose Complete Media without Epo||HSC004||3.0 mL||4.0 mL|
|Human Methylcellulose Enriched Media||HSC005||3.0 mL||4.0 mL|
|Human Methylcellulose Serum-Free Enriched Media||HSC005SF||3.0 mL||4.0 mL|
|Human Methylcellulose Serum-Free Enriched Media without Epo||HSC010SF||3.0 mL||4.0 mL|
|Methylcellulose Concentrate||HSC011||1.5 mL||2.25 mL|
|*Volume for Duplicate Experiments
**Volume for Triplicate Experiments
Note: When handling biohazardous materials such as human blood, safe laboratory procedures should be followed and protective clothing should be worn.
|Table 2. Determining the approximate cell number needed for each 35 mm culture dish.|
|Sample Source||Final Cell Number¹||Stock Cell Number (10x final)|
|Enriched CD34+ Cells||5.0 x 102 - 2.0 x 103||5.0 x 103 - 2.0 x 104|
|Cord Blood||5.0 x 103 - 2.5 x 104||5.0 x 104 - 2.5 x 105|
|Peripheral Blood||1.0 x 105 - 2.0 x 105||1.0 x 106 - 2.0 x 106|
|Mobilized Peripheral Blood||1.0 x 104 - 5.0 x 104||1.0 x 105 - 5.0 x 105|
|Bone Marrow (low-density)||1.0 x 104 - 5.0 x 104||1.0 x 105 - 5.0 x 105|
|¹ Final Cell Number per 35 mm Culture dish (or 1.1 mL 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.
|Table 3. Volumes necessary for experiments using 35 mm culture platees in duplicate or triplicate.|
|HSC001||HSC002, HSC002SF||HSC003, HSC004, HSC005,
|For experiments Using Cell
|Methylcellulose-based media||1.4 mL||2.1 mL||2.7 mL||3.6 mL||3.0 mL||4.0 mL||1.5 mL||2.25 mL|
|Culture supplements or cytokines||1.6 mL||2.4 mL||0.3 mL||0.4 mL||None*||None*||1.5 mL||2.25 mL|
|Cells||0.3 mL||0.45 mL||0.3 mL||0.4 mL||0.3 mL||0.4 mL||0.3 mL||0.45 mL|
|*No additional culture supplements or cytokines are needed.|
Score the colonies at the end of the incubation period. Identify and count the individual colonies using an inverted microscope and a scoring grid.
Colonies consisting of at least 40 cells are counted (or the minimum cell count set by each laboratory).
|CFU-E (Colony forming unit-erythroid): Clonogenic progenitors that produce only one or two clusters with each cluster containing from 8 to approximately 100 hemoglobinized erythroblasts. It represents the more mature erythroid progenitors that have less proliferative capacity.||CFU-G (Colony forming unit-granulocyte): Clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils or neutrophils.||CFU-GM (Colony forming unit-granulocyte, macrophage): Progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. The morphology is similar to the CFU-M and CFU-G descriptions.|
|BFU-E (Burst forming unit-erythroid): The size of the colony can be described as small (3 to 8 clusters), intermediate (9 to 16 clusters), or large (more than 16 clusters) according to the number of clusters present. These are primitive erythroid progenitors that have high proliferative capacity.||CFU-M (Colony forming unit-macrophage): Clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages.||CFU-GEMM (Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte): Multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages, as the name indicates.|
|Table 4. Colonies Supported by Methylcellulose Products.|
|Product Name||Catalog #||Colonies Supported|
|Methylcellulose Stock Solution||HSC001*||NA*||NA*||NA*||NA*||NA*||NA*|
|Human Methylcellulose Base Media||HSC002*||NA*||NA*||NA*||NA*||NA*||NA*|
|Human Methylcellulose Serum-Free Base Media||HSC002SF*||NA*||NA*||NA*||NA*||NA*||NA*|
|Human Methylcellulose Complete Media||HSC003||Yes||Yes||Yes||Yes||Yes||Yes|
|Human Methylcellulose Complete Media without Epo||HSC004||No||No||Yes||Yes||Yes||No|
|Human Methylcellulose Enriched Media||HSC005||Yes||Yes||Yes||Yes||Yes||Yes|
|Human Methylcellulose Serum-Free Enriched Media||HSC005SF||Yes||Yes||Yes||Yes||Yes||Yes|
|Human Methylcellulose Serum-Free Enriched Media without Epo||HSC010SF||No||No||Yes||Yes||Yes||No|
|*HSC001, HSC002, HSC002SF, and HSC011 do not contain any cytokines and will not support colony growth unless conditioned media, cytokines, or other culture supplements are added.|
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