Calcein red orange am

For these experiments we used GFP-expressing GSCs. Initially, HUVECs have been collected and resuspended in an appropriate volume and stained with Calcein Red-Orange AM (Invitrogen). Cells were plated on coverslips in growth medium for 24 h in the presence or absence of necrotic extracts (1 µg/mL). Meanwhile, GFP-GSCs have been treated with digoxin, or acriflavine in normoxic and hypoxic conditions for 24 h. After this time, GFP-GSCs have been collected and plated in the same wells with HUVEC for 24 h. Then, cells have been collected and washed. Glasses have been washed twice and then mounted on microscope slides. Fluorescence was observed by LSM 510 confocal microscope (Zeiss).

For live HCA microscopy experiments, 8,000 neuroblastoma cells were plated per well in 96-well plates (Grenier, Austria) in full supplemented DMEM and incubated overnight at 37°C, and 5% CO2 before starting neuronal differentiation with RA as described above. For live staining, culture media was removed and replaced with fluorescent dyes mix containing Hoechst 33342 (Merck-Sigma, United States) 1:10,000, CellTrace™ Calcein Green AM (Invitrogen, United States) 1:5,000 or Calcein Red-Orange AM (Invitrogen, United States) 1:5,000, diluted in HBSS. After 30 min incubation at 37°C, and 5% CO2 the plate was transferred to the IN Cell Analyzer 2,200 (GE Healthcare) for image acquisition under cell culture environmental conditions. Twenty images per fluorescent channel (fixed spacing fields) for each well were acquired in two different channels in less than 60 min. All images under a ×20 magnification were taken using the same acquisition protocol with constant exposures for each fluorescent channel. The multiple cell images were subsequently segmented and high content analyzed using IN Carta^® Image Analysis Software. Two dimension principal component analysis (PCA) was applied based on morphological features, followed by a ranking of features contributing to the separation in the PCA analysis.

All blood specimens from patients were obtained with informed consent according to the institutional review board-approved study protocol at the University of California - San Francisco (Study #21–35147), see Table S2 for demographic information. Fresh samples of peripheral blood from healthy adult volunteers were collected via a 23-gauge butterfly needle collection set (BD #23–021-022) into 10 ml Vacutainer EDTA tubes (BD #366643). Blood was kept on a shaker at minimum setting and utilized within 2 hours of the draw. Neutrophils were isolated using the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Tech #19666) with the BigEasy magnet (STEMCELL Tech #18001) according to the manufacturer’s protocol.
Isolated neutrophils were spun down at 200g for 5 min and resuspended in a dye media consisting of imaging media containing 5ug/ml Hoechst 3334 (Invitrogen #H3570) and 0.25 uM Calcein Red-Orange AM (Invitrogen #C34851). This cell suspension was incubated at room temperature in the dark for 15 min, and then the cells were spun down at 200g for 5 min. The dye medium was aspirated and replaced with R10 to achieve a final cell density at or below 1×10⁶ cells/mL. Purified neutrophils were then kept in polystyrene T25 flasks (Corning) at 37°C in a 5% CO2 environment until imaging. Cells were used ~5–8 hours post-isolation.

Fluorophores calcein AM (5–10 μM), Calcein Red-Orange AM (5 μM), LTR (100nM), Fluo-4 (10 μM), Alexa-488–phalloidin, and Rhodamine-phalloidin were purchased from Invitrogen. Human recombinant TNF-α (10–100 ng/mL, BD Biosciences), and BAPTA-AM (100 μM, Invitrogen) were used, as well as FK506 (100 μM), LPS (1–10 mg/kg), cytocholasin D (100 nM), jasplakinolide (100 nM), latrunculin B (50 nM), TB (0.01%), DTT (1 mM), Turk’s solution, and TB, which were purchased from Sigma-Aldrich. Protein A/G-agarose beads were purchased from Santa Cruz Biotechnolgoy Inc. Saponin was purchased from Calbiochem. EZ-Link N-hydroxysuccinimide-SS-biotin (catalog 21331) and streptavidin-Sepharose beads (catalog 20357) were purchased from Thermo Fisher Scientific.