Annexin 5 buffer

Caspase 3/7 enzyme activities were measured by using Caspase-Glo 3/7 Assay Systems (Promega, #G8090) according to manufacturer’s protocol. Cells were incubated with indicated compounds for 24 h, and the luciferase activities were measured after substrate incubation with a TECAN Infinite plate reader. Apoptosis assay was carried out using annexin V-APC (Thermo Fisher, #A35110) and propidium iodide (PI, BD Biosciences, #556463) according to manufacturer’s protocol. In brief, harvested cells (0.2 × 10⁶) were washed with PBS followed by annexin V buffer (Thermo Fisher, #V13246), and stained with Annexin V/PI mixture for 15 min at 25 °C. Cell cycle fractions were determined through PI nuclear staining. In brief, harvested cells (0.2 × 10⁶) were fixed with 70% cold ethanol, and stained with PI staining buffer (0.03 mg ml⁻¹ PI, 0.1 mg ml⁻¹ RNAse A, 0.1% Triton-X in PBS) for 30 min at 25 °C. The staining profile was acquired (≥10,000 cell events) using LSR II Flow Cytometer (BD Biosciences). The acquisition was analyzed using the FlowJo 8.1.1 software (Tree Star).

Spent media from 0 or 10 mM NH₄Cl-treated C6 cells were briefly centrifuged for 5 min at 4 °C and 3000× g to remove cell debris, before storage at −80 °C to await analysis. These supernatants were thawed on ice, and then centrifuged at 4 °C and 17,000× g for 15 min, before being resuspended in either serum free media (for stimulating GPNT cells), or Annexin V buffer (Thermo Fisher Scientific, Dartford, UK) for FACS flow cytometric analysis. EVs were stained with or without PE Cy7 Annexin V (to detect exposed phosphatidyl serine), from eBioscience (Thermo Fisher Scientific, Dartford, UK), as described previously [58 (link)]. Samples were acquired for 2 min on a FACS Canto II (BD Biosciences; Wokingham, Berkshire, UK), and EVs were counted by reference to enumeration beads (Flow count beads; Beckman Coulter, High Wycombe, UK). Data were normalised to facilitate pooling of multiple experiments (for presentation as fold increases); the range of total EV values for these normalised recordings varied from 549 to 2228 events, and whereas Cy7 annexin EVs ranged from 77 to 278 events.