CAMEO™-96 RES: Research

 

CAMEO Res logo



 

CAMEO™-96 Res

The only Standardized Colony-Forming Cell (CFC) Assay for Basic Research Applications

by

Combining the Power of Instrument-Based ATP Bioluminescence Proliferation Assays to Standardize the Colony-Forming Cell Differentiation Assay


INTRODUCTION
Since 1966, investigators have peered through a microscope to count colonies of primitive blood-forming cells. The colony-forming cell (CFC) assay has played an integral role in understanding the biology, physiology, organization and hierarchy of the lympho-hematopoietic system.

The CFC assay is a functional assay because the cell populations that it detects cannot be morphologically identified. As a result, the assay relies on the functional ability to produce colonies of morphologically identifiable cells in response to specific growth factors and/or cytokines, without which the cells would die. The ability to form colonies is made possible by the addition of a semi-solid viscous medium that allows the cells to grow, but restricts their movement within the culture. The newly divided cells therefore remain in place and form colonies. The number and type of colonies are counted under an inverted microscope. The colony number is dependent upon the number of cells inoculated into the culture and type and concentration of the growth factors and/or cytokines added. Prior to 1971, the semi-solid medium used was agar. Stephenson and colleagues used a plasma clot to immobilize cells of the erythropoietic lineage, while Iscove and coworkers developed the assay using water-soluble methylcellulose. The methylcellulose CFC assay is generally used to detect most primitive lympho-hematopoietic stem, progenitor and precursor populations.


DRAWBACKS OF THE CFC ASSAY
Despite its worldwide use in basic research, the colony-forming cell assay suffers from a number of drawbacks.

  1. Manual enumeration of colonies is highly subjective.
  2. There is a complete lack of standardization in colony enumeration procedures, despite the availability of colony atlases.
  3. Lineage and species comparisons are difficult to perform, as are studies involving large numbers of test compounds or other samples because of,
  4. Low-throughput of the assay procedure.
  5. Due to the lack of standardization and subjectivity of the manual enumeration process, a high degree of technical expertise is required involving extensive training.
  6. The assay cannot be validated.
  7. The assay is NOT a measure of direct proliferation potential; it detects differentiation potential. If detection of a proliferative response is required, the traditional colony-forming assay is NOT the assay of choice.

PROLIFERATION or DIFFERENTIATION? That is the question.
It has been assumed that the CFC assay measures proliferation, when it is actually measuring differentiation potential. In numerous publications, proliferation and differentiation are considered in the same “breath”, when results from the CFC assay are being described. Certainly proliferation and differentiation are, from a biological viewpoint, intricately intertwined. From an assay viewpoint however, it could not be farther from the truth.

The CFC assay is a differentiation assay. Manual colony enumeration relies on the colony-forming ability of the cells to differentiate and mature so that the colonies can be identified and counted. This is the basis of a functional differentiation assay whereby an unidentifiable and/or undifferentiated cell, e.g. a stem cell (by definition), acquires the features of a specialized cell. This is the definition of the differentiation process. As a result, stimulation of CFC-GEMM, for example, in a sample product using the CFC assay measures the differentiation potential of the CFC-GEMM to produce mixed colonies, erythroid, granulocyte-macrophage and perhaps even colonies of megakaryocytes. Even though proliferation is an inherent part of the process to produce colonies of cells, the CFC assay does NOT directly measure proliferation and is, therefore, NOT a proliferation assay.

Proliferation is defined as the expansion of cells by continuous division into initially two identical daughter cells. Proliferation therefore occurs prior to differentiation or a differentiation step. Without proliferation, differentiation would not occur. Differentiation is a default program requiring prior proliferation.

Even though proliferation and differentiation are part and parcel of lympho-hematopoietic system, these two processes have to be considered as separate entities as far as assays are concerned. There is no single assay that measures both proliferation and differentiation using the same readout.

Once proliferation and differentiation are considered completely separate entities with respect to assay measurement, it is then possible to understand and address how proliferation and differentiation can be measured for primitive lympho-hematopoietic cells.


CAMEO™-96 Res is the Answer
The CAMEO™-96 Res platform simultaneously combines a CFC assay with the power of an instrument-based ATP bioluminescence assay in the same culture.

In this assay system, both proliferation and differentiation are measured in the same culture, but using different readouts. The manual CFC assay procedure detects differentiation potential, while HALO® technology measures proliferation. In this way, investigators who still want to continue viewing and counting colonies to assess differentiation capability, can also measure proliferation and, more importantly, reap the benefits of a standardized CFC assay, that has hitherto been possible to perform.

When the CFC assay and HALO® are performed using the same reagents and under the same conditions, with both manual enumeration and measurement of bioluminescence performed on the same day, there is a direct correlation between total colony counts and the iATP concentration. This correlation produces a linear regression. It allows colony counts to be expressed as standardized iATP concentration (µM) equivalents. However, this correlation also provides a more important benefit, namely that the CFC assay can be standardized against HALO®. This means that for the first time, the CFC assay can actually be standardized and validated. Although basic researchers may not consider assay validation important for their studies, the ability to demonstrate this important assay characteristics is becoming increasing prevalent, especially when assays are used for commercial purposes.

It should be emphasized that even by including the manual CFC assay, the drawbacks and disadvantages associated with this assay procedure have not been eliminated, It is only by incorporating HALO® technology that has allowed for these improvements. Therefore, CAMEO™™-96 Res must be considered only as an interim solution to using alternative, instrument-based assays.


CAMEO™-96 Res Research Kits Catalog (This part of the Catalog has been included with the CAMEO™-96 STD)