Poly d lysine

The internalization assay was performed as previously reported^{43 (link)}. Briefly, HEK293 cells were transfected with plasmids encoding eGFP-tagged wild-type or mutant SSTR2. The transfected cells were plated on poly-D-lysine (Beyotime) coated coverslips in 24-well plates and grew overnight. The cells were washed with PBS, and then octreotide or paltusotine was added at the final concentration of 1–100 μM. After incubation for 30 min at 37 °C, the cells were fixed and permeabilized with methanol (−20 °C) for 5–10 min. Finally, the coverslips were mounted onto glass slides with the antifade mounting medium containing DAPI (Beyotime Biotechnology, P0131-5 ml). The confocal fluorescence images were aquired by Zeiss LSM 880 microscope with the ZEN imaging software (Zeiss).

ADSCs after five passages were exposed to a DCEF of 300 mV/mm in our laboratory-made equipment described in detail previously [12 (link), 13 (link)]. In brief, cells were seeded at 3 × 10⁴/cm² on a 100 mm petri dish, which was treated before seeding with poly-D-lysine (Beyotime, China) for 5 min and then left to dry for at least 30 min. Then, the cells were put back into the incubator for at least 6 h to achieve attachment. Coverslips were attached using high-vacuum silicone grease (Dow Corning, USA) to form a small chamber with silicone grease separating the petri dish into two reservoirs. Fresh medium was added into the petri dish, and the two separated reservoirs were allowed to connect. The petri dish was returned to the incubator for at least 12 h to allow for cell recovery. The medium was changed, and HEPES buffer (Solarbio, China) was added to reach 25 mM to maintain pH stability. Then, 2% agarose (Sigma, USA) salt bridges were placed on both sides of the petri dish, while the other side of salt bridges put in Steinberg's solution, connected with silver wires (Alfa Aesar, USA) to a direct current power supply (Maisheng, China). The voltage of the chamber was measured and adjusted every hour to reach the set chamber EF strength.

Neurobasal Medium, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), B-27 Serum-Free Supplement, L-glutamine and 0.25% trypsin-EDTA were purchased from Gibco, Langley, OK, USA. Cell culture flasks, 6-well plates, 96-well plates and Pasteur pipettes were purchased from Corning Inc., Corning, NY, USA. Deuterium oxide (D₂O), TMSP-2,2,3,3-D4 (D, 98%) and sodium-3-trimethylsilylpropionate (TSP) were purchased from Cambridge Isotope Laboratories, Tewksbury, MA, USA. D-(+)-glucose, streptozotocin (STZ) and ATP, ADP, AMP standards were purchased from Sigma Aldrich, St. Louis, MO, USA. Poly-D-lysine and the penicillin-streptomycin solution were purchased from Beyotime Biotechnology, China. The MAP2 antibody and goat anti-rabbit IgG-FITC antibody were obtained from Abcam, Britain and Santa Cruz Biotechnology, Santa Cruz, CA, USA, respectively.

The 120–160 g Wistar rats were killed by cervical dislocation and the ganglia was enzymatically dissociated to obtain the dorsal root ganglion (DRG) neuron cells. The operation procedures were investigated and approved by the Animal Ethics Committee of China Pharmaceutical University (SYXK(SU)2016-0011). Normal feeding occurred for at least three days for the animals to adapt to the environment, and we ensured that the mice had a free water supply during the experiment. As described in the literature [23 (link),35 (link)], the ganglia were rinsed in ice-cold Hank’s Balanced Salt Solution (HBSS) (Life Technologies, Carlsbad, CA, USA) and incubated in HBSS buffer containing 1.5 mg/mL type 2 collagenase (Sigma-Aldrich Inc., Milwaukee, WA, USA) at 37 °C for 30 min. Then, Dulbecco’s Modified Eagle’s Medium (DMED) (Gibco, Carlsbad, CA, USA), supplemented with 1% penicillin/streptomycin and 10% fetal calf fetal (Biological Industries, Kibbutz Beit Haemek, Israel) was used to rinse the ganglia three times and triturated with Pasteur pipettes. The DRG neuron cells were plated on a poly-D-lysine (Beyotime, Shanghai, China)/laminin (Sigma-Aldrich, Milwaukee, USA)-coated plate (Thermo Fisher, Massachusetts, MA, USA), incubated at 37 °C in 5% CO₂ at a relative humidity of 95%. All cells were used within 48 h.

Tumor cells were plated on sterile round glasses in 12 wells and incubated in nomoxia or hypoxia incubator for indicated times (24, 48, and 72 h). For another, sorted neutrophils subsets were seeded on Poly-D-lysine (0.1 mg/mL, Beyotime, #C0312) coated sterile round glasses at 12 wells in complete RPMI-1640 medium for 1–3 h (depending on the adhesion degree via optical microscope). For destroy NETs, DNAseI (Roche, #11284932001, 1000 Unit/mL) were added (Fig. S6C). Then the glass was fixed with 4% Paraformaldehyde (PFA) for 10 min at room temperature. Cell permeabilization was obtained after 20 min incubation with PBS containing 0.2% TritonX-100 (Sigma-Aldrich, #X100) and blocking by 3% BSA (MP Biomedicals, #0218054990) for 60 min. Cells were incubated with primary antibodies in 4 °C overnight and fluorescent secondary antibody was added then on the cells in 4 °C for 2 h. After washing, DAPI (Invitrogen, #D1306) was added and covered with glass. Samples were analyzed with LSM 710 confocal microscope (Carl Zeiss, Germany). Primary antibodies including HMGB1 (1:250, Abcam, #ab79823), neutrophil elatase (1:200, Abcam, #ab68672), Alpha Tubulin- Alexa Fluor® 488 (1:1000, Abcam, #ab7291) and Sytox Orange Nucleic Acid Stain (Thermo Fisher, #S11368) were applied.

HEK293T (ATCC, CRL-3216), HeLa (ATCC, CCL-2), and U2OS (ATCC, HTB-96) cells were cultured at 37 °C under 5% CO₂ in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, D6429) supplemented with 10% (v/v) FBS (PANSera, 2602-P130707) and 1% (v/v) penicillin/streptomycin (Gibco, 15140163). Cells were passaged every 2–3 days. For HEK293T transfection, 8 × 10⁴ cells were plated on 0.01% (w/v) poly-d-lysine (Beyotime, ST508) coated 24-well plate around 20 h before transfection. For HeLa and U2OS transfection, 6 × 10⁴ cells and 4 × 10⁴ cells were respectively plated on a 24-well plate around 20 h before transfection. Cells were transfected with 250 ng of each DdCBE monomer to make up 500 ng of total plasmid DNA using jetPRIME transfection reagent (Polyplus-transfection, 468 PT-114-75). Cells were harvested 72 h after transfection for genomic DNA extraction, then the region containing the targeted editing site was PCR amplified for Sanger sequencing (Azenta Life Sciences) or for producing libraries for next-generation sequencing.

L_4–6 DRG specimens were collected from different groups. In accordance with the experimental procedure of Batti et al. (2013) (link) and the improvement methods from the authors’ laboratory (Tan et al., 2020 (link)), the animals were sacrificed using 4% sevoflurane, with immediate stripping of L_4–6 DRG. Then, samples were cut with eye scissors, subjected to enzyme digestion, and centrifuged. After the clear liquid was removed, the specimens were placed on a 12-well plate that had been precoated with poly-D-lysine (Beyotime) for fixing cells. Then, N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) buffer was added and incubated at 37°C for 30 min, and the specimens were cleaned with a buffer. A fluorescence microscope was used to record the fluorescence intensity. When the concentration of Cl^– in the cell increased, the fluorescence intensity of MQAE was proportionally reduced.