Annexin 5 fitc and pi

Cells were seeded in triplicate in 96-well plates (1 × 10⁵ cells/ml) with or without different concentrations of inhibitor (SKP2 E3 Ligase Inhibitor C1 or NAE inhibitor MLN4924; Merck Millipore). Cell viability was assessed using AlamarBlue (ThermoScientific), according to the manufacturer's instructions, 48 h post-treatment.
For the evaluation of apoptosis, cells were stained with Annexin V-FITC and PI (Biolegend, London, UK) following manufacturer's instructions. A minimum of 20,000 cells were assayed for each condition on a flow cytometer (BD Canto), and analysed using FlowJo software (Tree Star, Switzerland).

A total of 5x10⁶ cells from un-induced or induced cultures were harvested at 300xg for 1min, washed once with PBS and resuspended in 1 ml of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, 10 mM glucose, pH: 7.4). Cells were then aliquoted in 200 μl and stained with FITC-annexin V and PI (BioLegend) according to the manufacturer's protocol. The samples were analyzed by FACS and visualized under a Nikon Eclipse E600 microscope.

The HL-60 neutrophil-like cell line was exposed to 1.5% DMSO for 6 days to induce differentiation into polymorphonuclear (PMN) cells. Differentiated cells were exposed to 600 μM H₂O₂ for 4 h to induce apoptosis. Concurrently, cells were treated with CM from BM, UC, and AD-MSCs, with and without cytomix pre-activation, or vehicle. Levels of apoptosis were determined using FITC Annexin V and PI (Biolegend, San Diego, CA, United States) on the BD Accuri^™ C6 Flow Cytometer and expressed as a percentage of total cells.

The solar simulated light was generated using a UV Solar Simulator UV SOL (Oriel, Newport Technologies, Irvine, CA, USA). The spectrum of light generated by the solar simulator consisted of 8.0% UVB and 92.0% UVA. The light was metered by measuring broadband UVB. The dose of emission was precisely regulated to be limited to UVA and UVB spectra (280–400 nm). Cells were pre-treated with 80 ng/mL OSM and vehicle for 1 h, irradiated using a solar simulator at the dose of 6 J/cm² (metered using broadband UVB). At 24 h post-irradiation, both floating and adherent cells were collected and stained with FITC Annexin V and PI (BioLegend). A negative control of unstained cells and positive controls of cells treated with 3% formaldehyde for 30 min on ice and stained with either PI or Annexin V were used to gate the flow cytometer for viable, early apoptotic, late apoptotic, and necrotic cell populations. Samples were subjected to FACS analyses (FACS Canto II, BD Bioscience). Data were obtained and analyzed using and FlowJo software (v.10.5.2, BD Bioscience).