Pronase e

The methods for the SCG neuron acquisition and whole-cell patch-clamp recording were described in our previous reports [7 (link),8 (link),9 ,10 (link)]. In short, SCG slices were cut, digested in the enzyme solution (50–60 min, 37 °C), and dispersed gently with glass tubes into a single neuron. For the enzyme solution, 0.6–0.7 g/L pronase E (Roche, Basel, Switzerland), 1.7–1.8 g/L collagenase type II (Worthington, Lakewood, CO, USA), and 7.0–8.0 g/L bovine serum albumin (Roche, Basel, Switzerland) were added to the incubation solution (NaCl 130 mmol/L, MgCl₂ 1 mmol/L, KCl 5 mmol/L, glucose 10 mmol/L, CaCl₂ 2 mmol/L, NaH₂PO₄ 1.5 mmol/L, NaHCO₃ 25 mmol/L, and HEPES 10 mmol/L). Here, we mainly recorded the characteristics of the delayed rectifier potassium channel current (I_K), sodium channel current (I_Na), and N-type calcium channel current (I_Ca). The data was recorded using Axopach 200B patch-clamp systems and analyzed using Clampex 10.2 software (Axon, Scottsdale, AZ, USA).

The method used followed the same procedures as those described by Schofield and Ikeda (1988) (link) and our previous studies (Li et al., 2010 (link); Cheng et al., 2016 (link)). Briefly, the rabbits were anesthetized, and the SCGs were removed and placed in the incubation solution (comprising NaCl 130 mmol/L, KCl 5 mmol/L, glucose 10 mmol/L, HEPES 10 mmol/L, NaH₂PO₄ 1.5 mmol/L, NaHCO₃ 25 mmol/L, MgCl₂ 1 mmol/L, and CaCl₂ 2 mmol/L, aerated with 95% O₂ + 5% CO₂ for 30 min, and adjusted to pH 7.3). Subsequently, SCG slices were cut and incubated for 30 min in the incubation solution (continuously bubbled with 95% O₂ + 5% CO₂). Afterward, SCG pieces were digested in another incubation solution (4 ml, adding 1.7–1.8 g/L collagenase type II (Worthington, United States), 0.6–0.7 g/L pronase E (Roche, Switzerland), and 7.0–8.0 g/L bovine serum albumin (Roche, Switzerland), 95% O₂ + 5% CO₂ for 50–60 min, 37°C). After digestion, SCG pieces were washed with the incubation solution and neurons were dispersed gently by glass tubes with small calibers. The neuron suspension was transferred into a culture dish for the following experiments.

The fresh in-situ HCC xenograft tissues were perfused at a flow rate of 10 mL/min with Gey's balanced salt solution (GBSS) for 10 min, followed by 100 mL of 0.12% pronase E (Roche) dissolved in GBSS for another 10 min [19 (link), 30 (link)]. The HCC tissues were then excised, dissected and incubated for 30 min with continuous shaking, with 0.04% pronase E, 0.05% collagenase and 0.002% DNase I (Sigma) in 100 mL GBSS. After digestion, the cell suspension was passed through a 0.22-μm mesh and centrifuged at 500 × g for 10 min. Subsequently, cells were purified with 8% Nycodenz (Sigma) gradient centrifugation. The resulting monocytes were then sorted by flow cytometry with anti-CD11c antibody (Abcam, Suzhou, China). Purified DCs were then generated via culturing in 6-well tissue-culture plates (Costar) plus 50 ng/mL GM-CSF and 10 ng/mL IL-4 (1,000 U/mL, R&D systems) for 5 days in RPMI 1640 FCS medium (no antibiotic). On day-5, DCs were determined by flow cytometry with anti-CD11c antibody (over 90% positive rate).

HSCs were isolated from BALB/c mice following the protocol published in 2015 [18 (link)] with some modifications (Figure 1A). Briefly, mouse was given intramuscular injections of 20 mg/kg of Ilium xylazil-20 (Troy Laboratories, Australia) and 14 mg/kg of Zoletil (Virbac, France) to induce deep anesthesia. Then, livers were digested by in situ perfusion with EGTA solution (Sigma–Aldrich, U.S.A.) for 2 min, pronase E (Merck, Germany), and collagenase D 0.038% for 5–7 min each (Roche Diagnostics, Germany). The mice were killed due to the change in circulation and the opening of diaphragm for liver perfusion. Next, the liver was excised into small pieces and further digested with pronase E and collagenase D supplemented with DNase I 1% (Roche Diagnostics, Germany) in vitro for 15 min. The digested solutions were then filtered through a 70-μm cell strainer and subjected to low-speed centrifugation (50×g for 3 min) to discard pelleted hepatocytes. The single-cell suspension was divided equally into three 15-ml tubes, mixed with a solution of Nycodenz (Axis-Shield, U.K.) to reach the final concentrations of 8, 9.6, and 11% separately and centrifuged at 1400×g for 20 min. HSCs were collected from the white layer. The number of isolated cells and cell viability were determined by Trypan Blue staining (Sigma–Aldrich, U.S.A.).