Apoptosis / Necrosis
detected by bioluminescence using
HALO®, LUMENESC™ or LumiSTEM™
Apoptosis or distinguishing apoptosis from necrosis is required in a large number of research applications. Many of these involve deciphering the mechanism of action of an agent.
There are now many ways of detecting whether cells are in the process of, or have undergone programmed cell death or apoptosis. These include DNA fragmentation assays such as TUNEL and the presence of phosphatidylserine on the surface of cells using Annexin V. These procedures may also distinguish between apoptosis and necrosis. However, they have to be used as stand-alone procedures and cannot be combined with cytotoxicity assays.
The initiation of apoptosis requires cellular energy in the form of ATP. Since HALO®, LUMENESC™ and LumiSTEM™ Platforms are based on the detection of changes in intracellular ATP which acts as a limiting substrate for a luciferin/luciferase reaction, the same assay that detects cytotoxicity can be slightly modified to detect apoptosis/necrosis in the same culture wells.
To detect apoptosis/necrosis, cell cultures are prepared in the normal manner. When the plates are processed, instead of taking a single luminescence reading after adding the luminescence reagent (reading A), the plate is left in the luminometer to detect the decrease and nadir of the iATP concentration. This is best performed using kinetic measurements usually available with the plate luminometer software. At this point, the ATP has been converted to ADP. The HALO® Apoptosis Kits contain an additional reagent that converts the ADP back into ATP. The time at which this converting reagent is added is noted (reading B). As the ADP is converted back into ATP, there is an increase in luminescence followed by a plateau in the luminescence readings. The last reading (reading C) then allows the ADP:ATP ratio to be calculated and therefore apoptosis. The form of the time course will also indicate whether apoptosis or necrosis has occurred.
