Developmental and Experimental Lympho-Hemotopoiesis

HALO® - The Colony-Forming Assay Alternative

For more than 4 decades, the developmental and experimental hematology community has used the Colony-Forming Assay (CFA). The CFA is a functional, differentiation assay, in that it detects the functional ability of a specific cell population, stimulated with specific growth factors and/or cytokines to produce colonies of cells that can be identified under the microscope. Since the production and differentiation of cells in the colonies relies on the growth factors and/or cytokines to induce the proliferation process, it is assumed and correctly so, that proliferation has taken place. However, the assay does not directly measure proliferation, because it does not detect a biochemical process that is proliferation dependent. Therefore the CFA is NOT a proliferation assay.

In 2002, HemoGenix® developed the HALO® Platform, a Colony-Forming Assay alternative. Originally designed using similar components to the traditional CFA, including methyl cellulose (MeC), the HALO® Platform does not suffer from the numerous drawbacks inherent in the CFA procedure. The most important drawbacks of the CFA procedure are subjectivity and the complete lack of standardization that does not allow experiments performed from day to day or over longer periods of time, to be compared directly with each other. Since all HALO® Platforms measure changes in intracellular ATP (iATP) as a biochemical marker for the proliferative process, detection is non-subjective because it is instrument-based, and standardized because it uses an external ATP standard.

As a result, virtually any of the studies that have previously employed the CFA procedure, can now be performed using a HALO® Platform to produce results that are non-subjective and fully standardized.

HemoGenix® has developed 2 HALO® Platforms, one that uses methyl cellulose to grow lympho-hematopoietic cells under clonal conditions (HALO®-96 MeC), and one that does not use methyl cellulose, but rather expands cells in suspension culture conditions (HALO®-96 SEC). The following table shows the difference between HALO®-96 MeC and HALO®-96 SEC as colony-forming assay alternatives for research.

Property

Colony-Forming

Assay

HALO®-96 MeC
HALO®-96 SEC
Type of assay:Differentiation

Proliferation / Cytotoxicity -
Apoptosis

 Proliferation / Cytotoxicity -
Apoptosis
Validated
No
Yes
Yes
Type of culture:
Methyl cellulose
Methyl cellulose
Suspension
Cell growth:
Clonal
Clonal
Expansion
Parameter measured:
Cell colonies
Intracellular ATP
 Intracellular ATP
Readout:
Manual / Microscopy
Luminescence / Instrument-based
 Luminescence / Instrument-based
Subjectivity:
Subjective
Non-subjective
Non-subjective
Standardization:
Not standardized
External ATP
External ATP
Format
35mm Petri dishes
96-well plate
96-well plate
Assay description:
Macro
Mini
Mini
Volume of assay:
1ml
100µl/well
100µl/well
No. of replicates:
2-3
Min. 4 - unlimited
Min. 4 - unlimited
Cell incubation time:
7-14 days
5-7 days
4-6 days
Turnaround time:
7-14 days
5-7 days
4-6 days
Processing time / Readout time:
None / ~10 min/plate 20 min/plate / <5min/plate
20 min/plate / <5min/plate
Throughput capability:
Low
Medium
Medium - High
Training:
6-12 months
2 days
2 days

Both HALO® Assay Kit Platforms are ideal for research purposes; in fact, HemoGenix® has developed a special product line just for the research scientist. HALO® Research Assay kits are available in the following 96-well plate configurations:

  • 1, 2, 3 or 4 plate kits.
  • All kits are very easy to use, have a very high sensitivity and are rapid, reliable and reproducible.
  • All HALO® Research Kits come with 96-well plates that have a transparent growth surface allowing you to view cell growth and, if necessary, count clusters and/or colonies. But once you've used HALO®, you won't want to count colonies again!!!
  • Kits are available without any growth factors, allowing you to add your own in any combination and at any concentration.
  • At least 12 different lympho-hematopoietic cell populations can be detected, including HPP-SP, CFC-GEMM, BFU-E, CFU-E, GM-CFC, G-CFC, M-CFC, Eo-CFC, Baso-CFC, Mk-CFC, T-CFC and B-CFC.
  • Many of these cell populations can be detected in combination in side-by-side assays.
  • All of these populations can be detected for multiple tissues, including bone marrow, peripheral blood, spleen, fetal and embryonic tissue.
  • Kits are available to detect cell populations of human, non-human primate, dog, rat and mouse either individually or in side-by-side assay combinations.
  • HALO® Kits are also available to detect apoptosis or distinguish apoptosis from necrosis.
  • Combine HALO® with phenotypic analysis or even gene expression analysis.
  • All kits include culture reagents, 96-well plates optimized for the culture reagents, standard ATP and high and low controls, reagents to detect luminescence and a detailed instruction manual that also provides information on how to setup your plate luminometer for automated results.

Try HALO® - The Colony-Forming Assay Alternative

without counting colonies

for all your cellular research needs