Calcein am cell permeant dye

Sodium L-ascorbate and alpha-lipoic acid were obtained from Sigma-Aldrich. The catalase activity assay was purchased from Abcam. The catalase antibody was obtained from Santa Cruz. The PrestoBlue™ BCA protein assay kit cell viability reagent and calcein, AM, cell-permeant dye were obtained from ThermoFisher. Other reagents used in the current study have been purchased from ThermoFisher.
Ascorbate (AA) and sodium alpha lipoate (LA, sodium salt of alpha-lipoic acid) were prepared freshly for each assay. The preparation of sodium alpha lipoate followed previous reports [25 (link)]. Briefly, lipoic acid and sodium bicarbonate were mixed equally and the solution was lyophilized to recover sodium alpha lipoate. The concentration of solution was determined by the UV absorbance at 330 nm.

Cells were seeded at 1 × 10⁴ cells per well in a 96-well plate and left to adhere overnight at 37°C. Cells were treated with the various reagents, washed with PBS, and analysed for cell viability using either PrestoBlue resazurin-based (Thermo Fisher Scientific) or CellTiter-Glo ATP-based (Promega) cell viability reagents according to the manufacturer’s instructions.
For live-cell imaging, cells were seeded in glass-bottom black opaque–walled 96-well plates and left to adhere overnight. Cells were stained with 1 μM calcein AM cell-permeant dye (Thermo Fisher Scientific) for 2 h at 37°C and replaced with 90 μl serum-free medium. 96-well plates were placed under a Zeiss Axio Observer inverted fluorescence microscope and positioned for imaging. A baseline t = 1 s image was made. Subsequently, at the same time repetitive imaging was started by one person making one image every second for 40 consecutive seconds, whereas another person added 10 μl of 10 times concentrated therapy to the well without interference with the set-up. Images were analysed and quantified using ImageJ software.

Purified NK cells were treated with ALT-803 (6.25, 12.5, and 25 ng/mL) or vehicle control (RPMI-1640 medium supplemented with l-glutamine, 10% FBS, and antibiotics) for 48 h before being used as effectors. On the day of the assay, human carcinoma cell lines (target cells) were labeled with 10 μM Calcein AM cell-permeant dye (Thermo Fisher Scientific) for 30 min and then seeded in triplicate at 3.0 × 10^{3 (link)} cells/well into black-walled flat-bottom 96-well culture plates (655090; Greiner Bio-One, Kremsmünster, Austria). Target cells were then treated with human IgG1 isotype control antibody (Thermo Fisher Scientific) or NEO-201 (dose range 0.1–10 μg/mL), and then, purified NK cells were added at effector-to-target (E:T) ratios of 6.25:1 and 12.5:1.
For blocking studies, purified NK cells were incubated at 37°C for 2 h with 15 μg/mL of antihuman CD16-neutralizing mAb (eBioscience, San Diego, CA) before being used as effectors. After 4 h of incubation at 37°C, 10 μg/mL the propidium iodide (Thermo Fisher Scientific) was added to each well and the plate was imaged and analyzed using the Celigo Imaging Cytometer (Nexcelom Bioscience LLC, Lawrence, MA). Specific ADCC lysis was calculated using the following formula: % specific lysis = 100 − [(average live target count experimental/average live target count control) × 100].

Cyto-spinned organoids (> passage 5) or cells were placed on glass slides, fixed in 4% neutral buffered formalin for 1 h, dehydrated, and permeabilized with 0.5% Triton at 4 °C for 1 min. Slides were blocked in 5% BSA in PBS and incubated with primary antibodies against P2XR4 1:100 PAE-83466 (Invitrogen), CD10 (DAKO Omnis; Clone 56C6), or Ki67 (Dako Omnis; Clone MIB-1) CAIX (H-11 Santa Cruz), and secondary antibodies, both at 4 °C overnight. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Caspase 3 activity was measured using the CaspGLOW™ Fluorescein kit in active caspase-3 (K183-100-N;Nexcelom), and 10 μM Calcein-AM cell-permeant dye (C1430 Thermofisher) was used to stain for cell vitality. Immunofluorescence images were acquired using a confocal Zeiss microscope and analyzed by ZEN2 program. MVs were prepared as previously described [19 (link)]. Particle diameters of the EV fractions in the range of 0–1000 nm were analyzed using a Zetasizer Nano ZSP (Malvern Panalytical, Malvern, UK).