CAMEO™-96
A Proliferation and Differentiation Assay for
Lympho-Hematopoietic Stem and Progenitor Cells
Combining a Colony-Forming Cell (CFC) assay with the power of an instrument-based, ATP Bioluminescence Readout to produce a proliferation and differentiation assay performed on the same sample from lympho-hematopoietic cells.
The new Online Catalog for CAMEO™-96 will be available soon
Background
The in vitro Colony-Forming Cell (CFC) assay was first published in 1966 independently by Bradley and Metcalf in Melbourne, Australia and Pluznik and Sachs in Rehovot, Israel. The original assay used agar as the semi-solid medium in which cells were stimulated with a conditioned medium that contained granulocyte-macrophage colony-stimulating factor (GM-CSF). The colonies obtained contained cells that could be identified as being part of the granulocyte-macrophage cell lineages. The cells producing these colonies were designated CFU (colony-forming unit) or CFU-C (colony-forming unit - culture), but are now generally termed granulocyte-macrophage colony-forming cells (GM-CFC or CFC-GEM).
Since that time, CFC assays have been developed to detect numerous stem, progenitor and precursor cells of the lympho-hematopoietic system. In 1973, Iscove and colleagues employed methyl cellulose as the semi-solid, viscous medium to all cells to grow into colonies that could be identified by the functionally, mature end cells produced. Methyl cellulose is now used for most of the cell populations detected using the CFC assay.
As mentioned, the identification of the colonies produced in the CFC assay is dependent on the production of functionally, mature cells as well as the "form" the colonies take during growth. Since the assay is dependent upon the cells differentiating and maturing into end cells, the CFC assay is a differentiation status or potential assay.
Although proliferation can be assumed to occur and is required for the cells to produce colonies, the CFC assay is NOT a proliferation assay and the readout does not measure proliferation, but rather differentiation. To measure proliferation, a different readout is required because proliferation and differentiation cannot be measured using the same readout.
CAMEO™-96
To detect and measure both proliferation and differentiation in the same assay, two different readouts are required. CAMEO™-96 combines the in vitro methyl cellulose CFC differentiation assay with the power of a highly sensitive and reliable, instrument-based, ATP bioluminescence proliferation assay that was first developed for the HALO® Platform.
CAMEO™-96 is performed in a 96-well plate. The target cells, at the correct cell concentration are added to a CAMEO™-96 Master Mix (provided with the assay kit) and 0.1ml is added to 6 replicate wells. The cells are cultured for the desired amount of time. After incubation, the total number of colonies is manually counted in each well using an inverted microscope. The number of colonies produced represents the differentiation readout.
After counting the colonies, the wells are then processed to measure the intracellular ATP (iATP) concentration. The iATP concentration is directly proportional to the proliferation status of the target cells at that point in time. To process the plate, a single reagent containing both a lysis and luciferin/luciferase reagents is assed to each well. After 10min incubation, the amount of light (photons) produced as a result of the iATP, luciferin and luciferase reaction is measured in a plate luminometer. Prior to measuring the bioluminescence produced in the sample wells, an extrenal ATP standard curve is performed that calibrates and standardizes the proliferation assay. The procedure is shown below.
Advantages of CAMEO™-96
1. CAMEO™-96 is the only assay kit that can detect and measure both proliferation and differentiation in the same sample for lympho-hematopoietic stem and progenitor cell populations.
2. CAMEO™-96 is the only assay that can standardize the CFC assay. This is because, the ATP proliferation assay is calibrated and standardized against an external ATP standard. Because both the differentiation and proliferation assay stages are performed using the same reagents and the cells cultured under exactly the same conditions, the manual, subjective part of the assay that detects differentiation as the total number of colonies counted, can be correlated directly with the ATP proliferation assay as shown in the graph below. This graph also demonstrates how the number of colonies counted can be converted to standardized iATP concentrations.
3. CAMEO™-96 is the only assay that allows the manual and subjective CFC assay to be validated. Since the iATP assay can be calibrated and standardized against an external ATP standard, this assay can and has been validated. This allows the CFC assay, which has not be validated against any other assay, to now be validated against the iATP proliferation assay.
4. CAMEO™-96 can be performed for many different stem and progenitor cell populations from several different species.
Applications of CAMEO™-96
Many of the advantages of CAMEO™-96 noted above are also applications.
1. All applications where both proliferation and differentiation have to be measured on the same cell population from the same sample.
2. All areas of basic research, including in vitro and animal models.
3. Stem cell transplantation and cord blood banking.
4. To determine the effect of various agents on both proliferation and differentiation simultaneously.
3. To separate a proliferation process or effect from a differentiation process or effect.
What's in the CAMEO™-96 Assay Kit Box
The CAMEO™-96 Assay Kit contains everything required to perform the assay except the cells and the instrumentation.
1. CAMEO™-96 Master Mix containing methyl cellulose for for culturing a specific cell population.
2. ATP Monitoring Reagent.
3. ATP Standard.
4. ATP high and low controls for the ATP standard curve.
5. Sterile 96-well plates for cell culture.
6. Non-sterile 96-well plates for the ATP standard curve.
7. Sterile adhesive foils to cover the 96-well plates so that unused wells can remain sterile for future use.
8. Instruction manual.