Hmgb1

nEV size and counts were determined by a nanoparticle tracking system (NTA), NanoSight LM10 instrument (Malvern Instruments, Malvern, UK) with a 405nm laser-equipped sample chamber, as described previously (Tang et al, 2016 (link)). The samples were diluted in PBS to 10⁸-10⁹ particles/mL. Camera shutter speed, camera gain and software detection threshold were manually adjusted for optimal detection and kept consistent during all sample analyses. Each sample was analyzed three times with three recordings of 30 seconds each. The modes were reported for particle sizes due to the highly skewed distributions. The mean of the triplicate recordings was reported for size and concentration. Protein concentrations in nEVs were analyzed using commercial ELISA kits, apoptosis-linked gene 2-interacting protein X (ALIX) (Lifeome-Cusabio, Oceanside, CA), synaptophysin (SYN) (American Research Products, Inc., Waltham, MA), HMGB1 (LifeSpanBioSciences, Inc., Seattle, WA), NFL (MyBioSource, San Diego, CA) and p-T181-tau (Fujirebio US, Malvern, PA). ELISAs were performed per manufacturer’s instructions and normalized to ALIX as previously described (Sun et al, 2019 (link)). All standards and samples were assayed in duplicate. Plates were analyzed using a SpectraMax M5 plate reader and SoftMax v5 software (Molecular Devices, San Jose, CA).

To quantify cytokines after ICD induction, each ICD induced tumor supernatants and DC-conditioned supernatants were collected after restimulation with lipopolysaccharide (LPS, 100 ng mL⁻¹) for 18 h. The concentration of HMGB-1 (Lifespan bioscience, USA), TNF-α, IL-12p70, and IL-10 was measured by ELISA (eBioscience, USA) with manufacturer’s protocol.