Nis elements br 4

For quantification of DNA-releasing ability in different samples, stained DNA entities were visualized by live imaging (4× objective). Three images were obtained from different positions in each well. Some degree of overlap occurred. The number of released DNA entities were counted using the software’s in-built object count feature (NIS Elements BR 4.20, Nikon). For quantification of DNA-releasing cells, cells were seeded in 150 μl PBS as described above and incubated for 30 min. Extracellular DNA was stained with SYTOX Green and visualized using live imaging. Multiple images were recorded using the Grab Large Image tool in NIS Elements BR 4.20 (Nikon) until the entire well was captured. Subsequently, the number of released DNA entities was manually counted and related to the number of cells seeded. Experiments were carried out three times for each condition at different occasions. When analyzing the impact of additional PBS washes on the ability to release DNA entities, Nalm-6 cells were washed in duplicates. Following the last wash, one sample was resuspended in PBS and used for quantification of released DNA entities while the other sample was resuspended in FBS containing PBS and used to determine the number of cells used in the quantification.

Scratch assays were performed using the Ibidi-culture inserts (Ibidi^®/Biovalley) following the manufacturer instructions. Cultures were then washed to remove detached cells and debris and treated with vehicle or 1 nM M4. Quantification of wound mean diameter were performed at t = 0 and after a 6 h exposure by Phase-contrast image analysis with NIS-elements BR 4.20.00 software (Nikon).

TUNEL assay was performed using the Apo-BrdU-IHC in-situ DNA fragmentation Assay kit (BioVision, USA) following the manufacturer instructions for the staining of cell preparations fixed on slides adapted for the use of AlexaFluor 555 (Invitrogen) goat anti-mouse secondary antibody. Nuclear DNA was stained with Hoechst (bisBenzimide H33342 Trihydrochloride, Sigma-Aldrich). Quantification of stained cell number was performed with NIS elements BR 4.20.00 software (Nikon).