Epiquik in situ histone h3 k9 acetylation assay kit

Cells were cultured in a 48 well plate (3 × 10⁴ cells/cm²) in basal medium. After 24 h, media was replaced with fresh basal medium and transwell inserts (0.4 µm pore size, Greiner Bio-One, Stonehouse, UK) containing EV-functionalised hydrogels, were placed into each well. Following 7 days of culture, the detection of H3K9 acetylation was performed using the EpiQuik^TM In Situ Histone H3-K9 Acetylation Assay Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer’s protocol. The absorbance was read in a SPARK spectrophotometer (TECAN, Männedorf Swizerland) at 450 nm. Histone acetylation was normalised with DNA content. Cells cultured with EV-free hydrogels were used as the control.

Cells were cultured in 96-well plates (3 × 10³ cells/cm²) in a basal medium for 24 h. The medium was replaced with a fresh basal medium supplemented with/without AZT (10 μM), DFO (10 μM) or AZT (10 μM)/DFO (10 μM) for 24 h, followed by 3 days of basal culture. The detection of H3K9 acetylation and methylation was performed using the EpiQuik^TM In Situ Histone H3-K9 Acetylation Assay Kit (Epigentek, New York, NY, USA) and EpiQuik^TM In Situ Histone H3-K9 Methylation Assay Kit (Epigentek, New York, NY, USA) according to the manufacturer’s protocol. The absorbance was read in a SPARK spectrophotometer at 450 nm. Histone acetylation and methylation was normalised with the DNA content.
The DNA quantification was determined by a Quant-iT PicoGreen DNA assay (Invitrogen, Life Technologies, Paisley, UK). Briefly, cells were lysed following three freeze-thaw cycles in 0.1% Triton^TM X-100 in phosphate-buffered saline (PBS, Lonza, Manchester, UK). A buffer of 90 μL of TE (10 mM Tris-HCl, 1 mM EDTA) was added to 10 μL of cell lysate in a 96-well plate (Corning, Deeside, UK). All samples received 100 μL of PicoGreen reagent and were incubated for 5 min. Subsequently, fluorescence was measured in a SPARK spectrophotometer at an excitation/emission wavelength of 480/520 nm.