CAMEO™-4 Platform

 

 

CAMEO Logo

 

Colony-Forming Cell Assays using the CAMEO™-4 Platform

 

Introduction

The original colony-forming assay (CFA) was published in 1966 independently by Bradley and Metcalf in Australia and Pluznik and Sachs in Israel. The assay was performed in agar and detected a population of cells that gave rise to both granulocytes and macrophages. This population, then called CFU-C or Colony-Forming Unit - Culture. This population is now usually called the granulocyte-macrophage colony-forming cell (GM-CFC or CFC-GM). In 1971, Axelrad and his colleagues used a plasma clot culture to detect erythropoietic precursor cells, designated colony-forming units - erythroid (CFU-E). Shortly afterwards, Iscove and his colleagues developed an equivalent assay, but using methylcellulose. The use of methylcellulose as a semi-solid immobilizing medium to grow colonies of primitive lympho-hematopoietic cell populations has now become the norm. In 1982, the assay, which had up to that time been performed as 1ml cultures in 35mm Petri dishes was miniaturized down to 100µl cultures. This miniaturization eventually became the HALO®™ Platform which can be performed in both 96- and 384-well plates. Despite the many drawbacks of the colony-forming assay technique, it is still used by many investigators today.

HemoGenix®® now provides the miniaturized version of the colony-forming cell assay in kit form. It is called CAMEO™-4 and can be used to detect up to 15 different lympho-hematopoietic cell populations from 5 species. The table below compares the normal colony-forming cell assay with CAMEO™-4.

The colony-forming cell assay has been used for numerous applications since the technique was first described. CAMEO™-4 (Colony Assay Miniaturization with Enumeration Output) is the first Colony-Forming Cell Assay to be produced and sold in this format. There are several advantages to using CAMEO™-4 instead of the traditional macro format. These are described below.

 

Characteristics of the CAMEO™-4 Colony-Forming Cell Assay Platform

The table below shows the differences between the traditional macro colony-forming assay technique and CAMEO™-4.

 


Colony-Forming Cell Assay:

(Macro Format)

CAMEO™-4

(Mini Format)

Type of Assay:
Differentiation
Differentiation
Type of culture:
Methylcellulose
Methylcellulose
Cell growth:
Clonal
Clonal
Parameter measured:
Cell colonies
Cell colonies
Readout:
Manual
Manual
Format:
35 mm Petri dishes
4-wells in 35 mm Petri dishes
Growth surface area:
8.7 cm2
0.97 cm2
No of replicates:
Usually 2-3 x 1ml each
4 x 100µl each
Volume of each assay:
1 ml
100 µl
Standardization:
Not possible
Possible when performed with HALO®

 

Cell Populations Detected using CAMEO™-4

CAMEO™-4 Kits can be obtained to detect each of the following 15 cell populations.

  • No growth factors (used for background control or to add growth factors and/or cytokines supplied by the investigator
  • HPP-SP “priming” (High Proliferative Potential Stem and Progenitor Cells)
  • HPP-SP “fully stimulated”
  • CFC-GEMM 1 (Colony-Forming Cells - Granulocyte, Erythroid, Macrophage, Megakaryocyte) .
  • CFC-GEMM 2 .
  • CFC-GEMM 3 .
  • BFU-E 1 (Burst-Forming Unit - Erythroid)
  • BFU-E 2
  • CFU-E
  • GM-CFC 1 (Granulocyte-Macrophage Clony-Forming Cell)
  • GM-CFC 2
  • G-CFC (Granulocyte Colony-Forming Cell)
  • M-CFC (Macrophage Colony-Forming Cell)
  • Mk-CFC (Megakaryocyte Colony-Forming Cell)
  • T-CFC (T-Lymphocyte Colony-Forming Cell)
  • B-CFC (B-Lymphocyte Colony-Forming Cell).

 

Growth Factor and Cytokine Combinations used to stimulate individual cell populations

  • HPP-SP 1 “Priming”: IL-3, IL-6, SCF, Flt3-L
  • HPP-SP 2 “Full stimulation”: EPO, GM-CSF, G-CSF*, IL-3, IL-6, SCF, TPO, Flt3-L, IL2, IL-7
  • CFC-GEMM 1: EPO, GM-CSF, G-CSF, IL-3, IL-6, SCF
  • CFC-GEMM 2: EPO, GM-CSF, G-CSF, IL-3, IL-6, SCF, TPO
  • CFC-GEMM 3: EPO, GM-CSF, G-CSF, IL-3, IL-6, SCF, TPO, Flt3-L.
  • BFU-E 1: EPO
  • BFU-E 2: EPO, IL-3, SCF
  • CFU-E: EPO
  • GM-CFC 1: GM-CSF
  • GM-CFC 2: GM-CSF, IL-3, SCF
  • G-CFC: G-CSF
  • M-CFC: M-CSF
  • Mk-CFC: TPO, IL3, SCF
  • T-CFC: IL2
  • B-CFC: IL-7

 

* Only included in kits to detect human cell populations

Species-Specific CAMEO™-4 Kits for Cell Populations

  • Human
  • Non-human primate
  • Dog
  • Rat
  • Mouse

Where possible, species-specific growth factors and cytokines are incorporated into the CAMEO™-4 kits.

 

What's in the CAMEO™-4 Kit Box?

All CAMEO™-4 Kits come with 2 x 15 ml bottles of CAMEO™-4 Master Mix and 50 x 4-well Petri dishes and manual. Please note that no cells or tissues are provided by HemoGenix®.

 

Requirements for performing CAMEO™-4

Other than the instruments and supplies normally used for tissue culture work there are no special requirements to perform CAMEO™-4. However, the use of syringes and needles to dispense reagents should not be used since they are not accurate and can lead to extremely high coefficients of variation. We strongly recommend the use of repeater pipettes (either manual or electronic) to dispense the methylcellulose-based CAMEO™-4 Master Mix and for plating the reagents.

 

How to use CAMEO™-4 Colony-Forming Cell Assays

The following protocol shows you how easy it is to use CAMEO™-4 for any cell population from any species.

 

CAMEO

 

 

CAMEO™-96 STD

Coming soon.

 

References in which the Miniaturization of the Colony-Forming Cell Assay has been used.

  1. The effect of reduced oxygen tension on colony formation of erythropoietic cells in vitro. Rich IN & Kubanek B. Brit. J. Haematol. (1982), 52:579-588.
  2. A role for the macrophage in normal hemopoiesis: I. Functional capacity of bone arrow macrophages to release hemopoietic growth factors. Rich IN. Exptl. Hemat. (1986), 8:738-745.
  3. A role for the macrophage in normal hemopoiesis: II. Effect of varying oxygen tensions on the release of hemopoietic growth factors from bone marrow-derived macrophages in vitro. Rich IN. Exptl. Hemat. (1986), 8:746-751.
  4. The effect of 5-Fluorouracil on erythropoiesis. Rich IN. Blood (1991), 77:1164-1170.
  5. The developmental biology of murine hemopoiesis: Effect of growth factors on colony formation by embryonic cells. Rich IN. Exp Hemat (1992), 20:368-370.
  6. Primordial germ cells are capable of producing cells of the hemopoietic system in vitro. Rich IN. Blood 86:463-472 (1995).

 

Technical Assistance

HemoGenix® is available to answer all questions regarding applications and technical assistance during normal work hours at no charge.