Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Nayan J. Sarma; Akiko Takeda; Nabeel R. Yaseen

doi:10.3791/2195

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

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11:30 min

December 18, 2010

DOI: 10.3791/2195-v

Nayan J. Sarma¹, Akiko Takeda¹, Nabeel R. Yaseen¹

¹Department of Pathology and Immunology,Washington University School of Medicine

Overview

The colony forming cell (CFC) assay is an in vitro method used to evaluate hematopoietic progenitors' ability to proliferate and differentiate in a semi-solid medium. This assay combines colony morphology, cell morphology, and flow cytometry to assess the hematopoietic lineages.

Key Study Components

Area of Science

Hematopoietic biology
Cell differentiation
In vitro assays

Background

The CFC assay is crucial for studying hematopoietic progenitor cells.
It helps in understanding the mechanisms of leukemogenesis.
Oncogenes' effects on hematopoietic differentiation can be assessed using this method.
Flow cytometry is employed to analyze cell populations.

Purpose of Study

To assess the proliferation and differentiation of hematopoietic progenitors.
To provide insights into hematopoietic lineage and maturation stages.
To evaluate the impact of cytokines and viral constructs on cell behavior.

Methods Used

Thawing CD34 positive cells and activating them with cytokines.
Retroviral transduction of GFP expressing constructs.
Isolation of GFP positive cells by flow cytometry.
Culture in semi-solid methylcellulose medium supplemented with growth factors.

Main Results

Colonies appear on the surface after approximately two weeks of incubation.
Harvested colonies can be analyzed for lineage and maturation stage.
Staining with specific dyes allows for microscopic evaluation.
The method reveals mechanistic insights into hematopoietic differentiation.

Conclusions

The CFC assay is a valuable tool for studying hematopoietic progenitor cells.
It aids in understanding the effects of oncogenes on differentiation.
This method can enhance research in leukemogenesis and related fields.

Frequently Asked Questions

What is the CFC assay?

The CFC assay evaluates the ability of hematopoietic progenitors to form colonies in a semi-solid medium.

How long does the CFC assay take?

The assay typically takes about two weeks for colonies to appear.

What are CD34 positive cells?

CD34 positive cells are hematopoietic progenitor cells that can differentiate into various blood cell types.

What role do cytokines play in the CFC assay?

Cytokines promote the activation and proliferation of hematopoietic progenitor cells.

How are the results of the CFC assay analyzed?

Results are analyzed through colony morphology, cell morphology, and flow cytometry.

What insights can be gained from the CFC assay?

The assay provides insights into hematopoietic differentiation and the effects of oncogenes.

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

This procedure assesses in a semi-solid medium, the ability of hematopoietic progenitors to proliferate and differentiate along different hematopoietic lineages to begin culture. Freshly thawed CD 34 positive cells in the presence of cytokines to promote activation after two days. Perform retroviral transduction of a GFP expressing test construct by adding the activated CD 34 positive cells to a plate preloaded with virus.

48 hours later isolate the GFP positive cells by fax, then plate these cells in semi-solid methylcellulose medium supplemented with growth factors incubate for approximately two weeks until colonies appear on the surface. Finally, harvest the colonies, immobilize the cells on slides using cytosine and stain with right giza for microscopic determination of hematopoietic lineage and maturation stage. This method can provide mechanistic insight in studies of leukemogenesis, namely the effects of oncogenes on hematopoietic differentiation.

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