Collagenase buffer

Primary hepatocytes were isolated by a two-step collagenase perfusion method according to a previous report [22 (link)] with modifications. After CAG-ELuc/MI-MAC Tc mouse was anesthetized with 3% isoflurane gas using small animal anesthetizer (MK-AT200, Muromachi Kikai, Tokyo, Japan), the liver was perfused with liver perfusion medium (Gibco-BRL) via the hepatic portal vein and immediately the abdominal inferior vena cava was cut to induce bleeding. After the liver had been freed of blood, liver perfusion medium (Gibco-BRL) was replaced with 0.5 mg/ml collagenase buffer (Sigma-Aldrich) for 4 min. The perfusion rate of 5 ml/min and the temperature of around 40 °C were maintained for both perfusates and collagenase buffer during the entire procedure. After perfusion, the liver was rapidly transferred to a sterile petri dish and shredded in cold hepatocyte wash medium (Gibco-BRL). The medium containing shredded liver was filtered through a 100 μm cell strainer (Becton Dickinson, Bedford, MA). After washing by low-speed centrifugation at 50 g for 3 min at 4 °C, the hepatocytes were suspended in the appropriate medium for subsequent experiments.

In the primary hepatocyte isolation experiment, anaesthetized mice were inserted with a catheter into the hepatic portal vein and perfused with Hank's Balanced Salt Solution (Invitrogen, CA, USA). After perfusion, the liver was perfused with collagenase buffer (0.375 μg/mL) (Sigma–Aldrich, Shanghai, China) five times. Then it was transferred to a dish, minced, filtered through a 75 μm mesh, and washed in DMEM three times to obtain hepatocytes. The hepatocytes were centrifuged in Percoll, resuspended in DMEM, stained with Trypan blue and counted. Isolated hepatocytes were plated into cell culture dishes and subsequent experiments were conducted 24 h later. AML12 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM/F-12 (1:1) supplemented with fetal bovine serum (10%), ITS liquid media supplement (1%) and dexamethasone (40 ng/mL). The cells were cultured in culture plates at 37 °C and 5% CO₂ in an incubator. In the experiments, hepatocytes were treated with Palmitic acid (0.125, 0.25, 0.5, 0.75 or 1 mM), leptin (25, 50 or 100 ng/mL) or Stattic (5, 10 or 20 μM). Palmitic acid was purchased from Sigma–Aldrich (Shanghai, China). Recombinant murine leptin was purchased from Peprotech (Suzhou, China). Stattic was purchased from Selleck (Houston, USA).

Tumor cells suspensions were obtained by enzymatic digestion with 270 units/ml DNase, 35 U/ml hyaluronidase, and 200 U/ml collagenase buffer (all from Sigma-Aldrich). After a 30-min incubation at 37°C, the cells were passed through a 70-µm cell strainer (BD Biosciences, San Jose, CA, USA). Samples were then stained at 4°C with a mouse anti-hPSMA mAb [19] (link) followed by a PE-conjugated anti-mouse IgG1 secondary antibody (BioLegend) and analyzed on a FACSCalibur (BD) flow cytometer. Data were evaluated with FlowJo software (TreeStar Inc., Olten, Switzerland).