How Does HALO® Work?

 

HALO Research Kit Logo

 

The HALO® Platform is an in vitro, non-invasive, cell-based proliferation assay. When lympho-hematopoietic cells proliferate or are inhibited from proliferating, the intracellular ATP (iATP) concentration changes proportionately. After incubation, the iATP is released from the cells by lysis. The iATP acts as a limiting substrate for a luciferin / luciferase reaction to produce bioluminescence in the form of light according to the following equation:


                                Luciferase
ATP + Luciferin + Oxygen -------> Oxyluciferin + AMP + PPi + CO2 + LIGHT

 

Characteristics of the new, 3rd Generation Luminescence Reagent


Simple
  • "Add-Mix-and-Measure" format uses a single reagent incorporating both cell lysis and luminescence-generating reagents.
Increased Sensitivity
  • 2-3 times brighter signal means signficantly increased sensitivity and reduced cell concentration requirement.
Fast
  • Only 15 minutes to develop and measure results in a 96-well plate.
  • Cuts time in half compared to previous reagents and saves on supplies.
Reagent Stability
  • Significantly reduced decay times.
  • No need to measure luminescence immediately.
  • Improved shelf life.
  • Improved handling and shipping conditions.

The bioluminescence produced is detected in a plate luminometer (required to perform HALO® as well as LUMENESC™ and LumiSTEM™). The luminometer readout is non-standardized Relative Luminescence Units (RLU). To convert RLU into a standardized ATP unit, a standard ATP dose response is performed prior to measuring the samples. The software used to drive the plate luminometer can usually be programmed to automatically convert RLU values into calibrated ATP (µM) values. A typical ATP dose response curve is shown below.

ATP Dose Response

Converting non-standardized RLU values into calibrated ATP values is an extremely important step in all bioluminescence measurements. By performing the ATP standard dose response, you are ensuring that both your luminometer and the luminescence reagents are functioning correctly.
 
By calibrating your results to the external ATP standard provided with all luminescence kits from HemoGenix® (HALO®, LUMENESC™ and LumiSTEM™), you can now compare results directly from one experiment to another regardless of the time span between the experiments. Furthermore, you can also compare results between multiple laboratories, thereby validating the assay for a specific procedure.
 
This is of paramount importance for the HALO® Stem and Progenitor Cell - Quality Control (SPC-QC) Platform, since stem cell transplantation and cord blood storage processing laboratories can use the HALO® SPC-QC Platform to determine standardized growth potential / potency of the processed product in a validated manner. The traditional colony-forming assay cannot be calibrated and therefore procedures cannot be standardized or compared amongst processing laboratories. For more information of the HALO® SPC-QC Platform, please click on the link.
 
LUMINOMETERS
A plate luminometer is not the same as that for measuring absorbence or fluorescence. Multi-parameter instruments are available that can measure, absorbence, fluorescence and luminescence. Alternatively, a dedicated luminometer that only detects luminescence (both chemi- and bioluminescence) can also be obtained.
 
At HemoGenix® we have tested a number of luminometers from different manufacturers. These include instruments from:
  • TECAN
  • Molecular Devices
  • BioTech
  • Bertold
Please call HemoGenix® at 719-264-6250 or e-mail HemoGenix® at info@hemogenix.com for advice and any other questions you might have regarding luminometers. HemoGenix® will also be marketing a luminometer in early 2007.