Trypsin edta 1x

MDA-MB-231 cancer cells (1.5 × 10⁶ cells) were cultured in a 6-well plate for 24 h and treated with DMSO as control or dihydroconiferyl ferulate (50 μM) for 24 h [63 (link)]. The cells were harvested, dissociated, and incubated with monoclonal antibody antihuman CD44 (FITC-conjugated) and monoclonal antibody antihuman CD24 (APC-conjugated) antibodies (BD) for 45 min at 4 °C. After washing with 1X PBS, the CD44⁺/CD24⁻ cell populations were examined using an Accuri C6 machine (BD San Jose, CA, USA). The Annexin V Apoptosis Detection kit with PI (BD) was used to measure apoptosis of the mammospheres treated with dihydroconiferyl ferulate (50 μM) following the manufacturer’s protocol. Mammospheres were collected and dissociated with 0.05% trypsin-EDTA 1X (Corning). Briefly, 1 × 10⁶ cells were incubated with Annexin V (FITC) and PI in a binding buffer at room temperature (RT), protected from light for 30 min, and the cells were examined via flow cytometry at the Jeju Center of Korea Basic Science Institute (core facility center, Jeju, South Korea). Mammospheres derived from MDA-MB-231 cells cultured with or without dihydroconiferyl ferulate (50 µM) were divided into single cells, and an equal number of cancer cells were seeded into six-well plates. The number of cells was counted at 24, 48, and 72 h to measure the growth of the mammospheres.

Tissue specimens obtained were washed in phosphate buffer saline (PBS) and processed within 2 h of surgical resection. Samples were washed with physiologic solution, minced with fine sterile scissors and scalpeled into fragments of approximately 1 mm³. Cells were immediately isolated from cirrhotic tissue proximal (1 < CP < 3 cm from the tumor resection margin) and distal (CD > 5 cm from the tumor resection margin or contra-lateral lobe) to the HCC lesion. The cell isolation procedure was performed as previously shown [42 (link)]. Briefly, the 1 mm³ fragments of tissue were incubated for 3 h with Fetal Bovine Serum (FBS) HyClone (Fisher Scientific, Hampton, NH, USA). FBS was then replaced by complete RPMI 1640 medium with 10% FBS, 1% of antibiotics (Corning Inc., Corning, NY, USA) and amino acids (Sigma-Aldrich, St. Louis, MO, USA; MEM Non-essential amino acid solution (100×) #M7145) and incubated for 24 h. Every 48 h cells were then washed with 2 mL of RPMI 1640 complete medium. After 8 weeks a monolayer of primary cells around the explants was observed. Cells were detached using Trypsin/EDTA 1X (Corning), re-plated, and maintained in culture at 37 °C and 5% CO₂.