5.2.6. PARP-1 Cleavage

PARP-1 is the enzyme present in the nucleus that is involved in DNA repair and is implicated in other essential cellular processes. When caspase-3 is activated during apoptosis, it cleaves PARP-1 (between Asp214 and Gly215), leading to the formation of two fragments of 85 kDa and 25 kDa. The addition of an HLNC4 antibody (conjugated with Alexa Fluor 488) allows the specific detection of the 85 kDa PARP-1 fragment.

The cells were treated with 2,4,6-TBP or PBP at 25 µg/mL and incubated for 24 h at 37 °C in total darkness. Then, PBMCs were washed and suspended in 1% paraformaldehyde (dissolved in PBS solution). Finally, an HLNC4 antibody conjugated with Alexa Fluor 488 was added to the samples that were incubated for 30 min at 37 °C in total darkness. In the cells, apoptosis was induced by camptothecin at 10 μM (positive control). A cytometric analysis of the samples was performed (LSR II, Becton Dickinson) at excitation/emission maxima of 494/519 nm for Alexa Fluor 488. The FMC gate on PBMCs was established, and the data were recorded for a total of 10,000 cells per sample.