Detecting Apoptosis / Necrosis with HALO®
There are many ways of detecting apoptosis, e.g. DNA fragmentation methods such as TUNEL, detection of phosphatidylserine on the surface of cells using Annexin V. However, when detecting cytotoxicity or any other agent or perturbation that can result in a decrease in proliferative activity, there are no assays, other than HALO® that can measure inhibition of proliferation and couple the detection system simultaneously with a measurement of apoptosis. This is because for apoptosis to be initiated, chemical energy in the form of ATP is required. Since HALO® detects changes in intracellular ATP that are directly proportional to the proliferative status of the cells being measured, a kinetic modification of the bioluminescence readout allows HALO® to detect apoptosis or necrosis.
Apoptosis can be detected using any of the HemoGenix® ATP-based bioluminescence detection systems. For HALO®, 3 separate platforms are available to detect proliferation / cytotoxicity and apoptosis. These are:
- HALO®-96 MeC (Methyl Cellulose),
- HALO®-96 SEC (Suspension Expansion Culture)
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. At this point, the ATP has been converted to ADP. The HALO® Apoptosis Kits contain an additional reagent that converts 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 the apoptosis. The form of the time course will also indicate whether apoptosis or necrosis has occurred.
