Low attachment 6 well plate

Huh7 cells were seeded in low-attachment 6-well plates (Corning, NY, USA) at a density of 3 × 10³ cells/well with or without 10 nM Taxol, 10 µM Cisplatin, or 0.2 µM CPO for 7 days. The stem cell permission media was composed of DMEM/F12 (Gibco) supplemented with 1× B27 (Invitrogen, Eugene, OR, USA), 20 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), 20 ng/mL epidermal growth factor (EGF; Invitrogen), and 25 µg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA). After incubation, the spheroids were observed using Operetta HCS system (Perkin Elmer, Waltham, MA, USA).

Melanoma cells (2.5 × 10⁵ cells) were seeded in a T-25 culture flask and allowed to adhere for 4 hours. Culture media was replaced and amended with media containing 100 μM Lunasin or vehicle for 24 h. Cells were harvested using enzyme-free trypsin (TrypLE, Life Technologies), counted, and subjected to labeling. For identifying ALDH^high cells, melanoma cells were assayed with ALDH as described above. ALDH^high and ALDH^low melanoma cells were sorted using a MoFlo cell sorter (Dako Cytomation). Sorted ALDH^high cells (1 × 10³ cells/mL) were plated in low-attachment 6-well plates (Corning) in DMEM/F-12 serum-free media. Cells were either treated with 100 μM Lunasin or vehicle and labelled with Annexin V/PI. Labelled cells were analyzed by flow cytometry using FACSCalibur (BD Biosciences).

Around d150, hCSs were transferred to low-attachment 6-well plates (Corning) and incubated with or without 100 ng/ml Lipopolysaccharide (LPS) (Sigma-Aldrich, L4391-1MG, dissolved in PBS, 2 ml total NSM per 6-well) for 24 h. Subsequently, LPS was washed off with DPBS and hCSs were incubated with or without 5 ng/ml Tumor necrosis factor alpha (TNFα) (Sigma-Aldrich, T0157-10UG, dissolved in PBS) and 5 ng/ml Interleukin-1 beta (IL-1β) (Sigma-Aldrich, SRP3083-10UG, dissolved in PBS) for 24 h. Spheroids were then harvested and processed as described below.

TS/A cells were electrotransfected with gWiz Blank plasmid as described above using EP1 and EP2 pulse protocols. The cells were then seeded into low-attachment 6-well plates (Corning Incorporated, Corning, NY, USA) and incubated for 9 hours. After incubation, the cells were lysed using RIPA buffer in accordance with the manufacturer’s protocol (Santa Cruz Biotechnology Inc., Dallas, TX, USA). For the preparation of crude lysate, the final centrifugation step was excluded. Total protein content was determined using a Pierce BCA Protein Assay Kit (Fisher Scientific) then adjusted with RIPA buffer. Twenty-five µg or 40 µg of total protein (for cleared and crude lysate, respectively) per well was separated in a 10% polyacrylamide gel, transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA), blocked for 1 hour in 5% milk in TBS Tween-20 buffer (Fisher Scientific) and incubated overnight at 4° C with primary antibodies: rabbit anti-DAI/ZBP1 or rabbit anti-β-actin as a loading control (Fisher Scientific). After washing with TBS Tween-20 buffer, the membrane was probed with Alexa Fluor 680 goat anti-rabbit secondary antibody (Fisher Scientific) for 45 min at room temperature, washed and protein bands were visualized with Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE, USA). The band intensity was quantified using Image J software [97 (link)].

For cytokine-stimulation experiments, d150 hCSs were transferred to low-attachment 6-well plates (Corning) that were placed on a gentle cell-culture incubator rocker in order to achieve an optimal flow of cytokines during the stimulation period. hCSs were incubated with 5 ng/mL TNFα (Sigma-Aldrich T0157-10UG; in PBS) or 5 ng/mL IL-1β (Sigma-Aldrich SRP3083-10UG; in PBS) for 4 h, 12 h or 36 h. The cytokine concentrations and stimulation time points were chosen based on our previous studies, studies utilizing 2D cell models [17 (link),18 (link),19 (link),20 (link),21 (link)], and the only study published thus far that dealt with cytokine (TNFα) stimulation of a 3D human brain organoid [22 (link)]. Following stimulation, spheroids were harvested for RNA isolation and the RNA was subjected to RNA-seq analysis as described below. Unstimulated hCSs from the same batch were used as controls. For pro-inflammatory stimulation experiments with HMC3 and HOG cells, the cells were incubated with 100 ng/mL LPS (Sigma-Aldrich, L4391-1MG) (dissolved in PBS) for 24 h (24 h) with subsequent incubation with 5 ng/mL TNFα (in PBS) and 5 ng/mL IL-1β (in PBS). Following stimulation, HMC3 and HOG cells were harvested for RNA isolation and the RNA was subjected to RNA-seq analysis, as described below.

Purified monocytes were cultured in RPMI1640 (Gibco or Lonza) medium containing 5% FCS (Corning, NY, USA), 1% Penicillin/Streptomycin (Gibco), and 10 ng/ml human recombinant M-CSF (Peprotech, London, UK) for their differentiation into macrophages. 3 x 10⁶ cells/well were seeded in low-attachment 6-well plates (Corning) and incubated for 7 days at 5% CO₂ and 37°C. Macrophages were collected and cultured in 24-well cell culture plates as 5 x 10⁵ macrophages/well for 24 hours in RPMI1640 medium containing 5% FCS, 1% Penicillin/Streptomycin. On the following day, the cell culture media was replaced with fresh media and macrophages were stimulated with the relevant polarization factors as described below. The cells were verified to be over 90% CD68⁺ by flow cytometry.