Proliferation & Apoptosis Assays
Evaluation of cell cycle progression is essential for investigations in many scientific fields. Measurement of [3H] thymidine incorporation as cells enter S phase has long been the traditional method for the detection of cell proliferation. Subsequent quantification of [3H] thymidine is performed by scintillation counting or autoradiography. This technology is slow, labor intensive and has several limitations including the handling and disposal of radioisotopes and the necessity of expensive equipment.
A well-established alternative to [3H] thymidine uptake has been demonstrated by numerous investigators. In these methods bromodeoxyuridine (BrdU), a thymidine analog replaces [3H] thymidine. BrdU is incorporated into newly synthesized DNA strands of actively proliferating cells. Following partial denaturation of double stranded DNA, BrdU is detected immunochemically allowing the assessment of the population of cells, which are actively synthesizing DNA.
Apoptosis, also known as programmed cell death or ankoikis, leads to the elimination of cells without releasing harmful substances into the surrounding area. Apoptosis is the result of a cascade of molecular and biochemical events involving endogenous endonucleases that cleave DNA into the prototypical ‘ladder of DNA fragments’. In addition to producing classical DNA ladders, the apoptotic endonucleases generate free 3’-OH groups at the ends of these DNA fragments to allow for the detection of apoptotic cells using a molecular biology-based, end-labeling technique.