Psiva

The synaptosome-containing pellet (above) was resuspended in binding buffer (10 mM HEPES pH 7.4), 150 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1.8 mM CaCl₂), after which 100 μl aliquots were incubated for 30 min at 20–25 °C with dye solution and then diluted in PBS (final volume, 1000 μl) for immediate flow cytometry analysis. Final dye concentration was 1 μM for Calcein AM Blue (Invitrogen) and 5 μl/ml for p-SIVA (Novus Bio.). The synaptosomes were analyzed on a BD FACSAria Cell Sorter.

After collection, aliquots (100 µL) of A549 media were dried under N₂ gas and prepared in LDS sample buffer for electrophoresis separation and membrane transfer. HMGB1 was detected via an HMGB1 rabbit pAb (ab79823) and antirabbit HRP secondary antibody and visualized with an ImageQuant LAS 4000 camera. HMGB2 was similarly detected via an HMGB2 rabbit pAb (ab124670) and antirabbit HRP. Western blots were developed with ECL reagents. Fluorescent staining for nuclear DNA, necrosis, and apoptosis was done with Hoechst 33342, pSIVA, and PI dyes (Novus, NBP2-29382), respectively, following washing of live cells with PBS. Fluorescent microscopy was performed with BioTek Gen5 3.04 Imager. Images were generated from overlaying the blue, green, and magenta filters, respectively.