Microplate reader

NKCs were labeled with CAM (Dojindo, Kumamoto) as described above. After incubation with blocking antibodies against SDF-1α/CXCL12 (Abcam) and MCP-1/CCL2 (Abcam) for 90 min at room temperature, the iPSC-CM sheet culture supernatants were placed in the bottom wells of a Transwell migration plate (Costar Corning). CAM-labeled NKCs were added to the upper wells, and plates were incubated for 90 min 37 °C to allow the cells to migrate. The fluorescence intensity was recorded with a microplate reader (DS Pharma Biomedical) with an excitation wavelength of 490 nm and an emission wavelength 520 nm. Experiments were performed in triplicate. The migrated cells were quantified by measuring the fluorescence intensity of the bottom wells, and the chemotaxis of NKCs was calculated as the percentage of the fluorescence intensity compared with that of the control well, in which NKCs were added to the bottom wells.

After splenocytes derived from C57BL/6 were obtained as described above, NKCs were isolated from splenocytes by magnetic-activated cell sorting using negative-selection kits (NK Cell Isolation Kit II, Miltenyi Biotec) and cultured using ALyS505NK-EX medium (Cell, Science & Technology Institute Inc.) with 1000 U/ml mouse IL-2 (BioLegend) for two weeks. iPSC-CMs were treated with 100 ng/ml IFN-γ (R&D Systems) for 48 h. LDH-release assays were performed using an LDH Cytotoxicity Detection Kit (Takara Bio). Cultured NKCs were washed and incubated with blocking antibodies against DNAM-1 (CD226) (Thermo Fisher Scientific) and NKG2D (BioLegend) for 90 min at room temperature. After washing, NKCs were cocultured with iPSC-CMs in a 96-well plate for 6 h. After incubation in the dark for 30 min at room temperature, the absorbance at 490 nm was recorded with a microplate reader (DS Pharma Biomedical), with a reference wavelength of 600 nm. The experiments were performed in triplicate. LDH release was determined as the percentage of LDH released compared with that of the 0.5% Triton X-treated control.

The LDH release assay was performed using LDH Cytotoxicity Detection Kit (Takara Bio, Shiga, Japan). FN-G-coated and uncoated hiPSC-CMs (2.5 × 10⁴ cells/well) were respectively seeded into 96-well plates and cultured at 37°C for 1 day. The next day, the media were changed, and the cells were incubated under hypoxia (5% O₂, 37°C) for 3 days.
A mixture of diaphorase and NAD⁺ was added to each well. After incubation in a dark room for 30 min at room temperature (20–25°C), the absorbance was recorded on a microplate reader (DS Pharma Biomedical, Osaka, Japan) at 490 nm with a reference wavelength of 600 nm. The experiments were performed in triplicates. LDH release was determined as the percentage of LDH release compared with that of the control.

A sandwich enzyme-linked immunosorbent assay (ELISA) was used for determination of alliinase protein as described by Kamata et al.37 . An antibody against alliinase was raised in rats by Operon Biotechnologies (Tokyo, Japan) using a purified onion alliinase protein. Affinity-purified anti-alliinase antibody was used as the capture layer in a sandwich ELISA. For the detection antibody, the anti-alliinase antibody was biotinylated using Biotin Labeling Kit-NH2 according to the manufacturer’s protocol (Dojindo Mol). A microplate reader (DS Pharma Biomedical) was used for the detection. The quantification range of the ELISA was 0.3–4.8 μg/mL of purified garlic alliinase protein as the standard. Alliinase protein expression levels are presented as the ratio relative to the total protein quantified using a Protein Assay Kit (Bio-Rad) with bovine gammaglobulin (SIGM) as the standard.

MCF-7 cells (2.5 × 10³/well) were seeded in 96-well plates and incubated for 24 h. Then, the cells were treated with different concentrations of antipsychotics and tamoxifen citrate. The medium was exchanged 48 h after seeding the cells. After treatment for 72 h, the cells were washed with PBS. Then, 100 μL 10% FBS-RPMI+ 10 μL WST-8 solution was added to each well and incubated for an additional 1 h at 37 °C with 5% CO₂. Absorbance at 450 nm was measured with a microplate reader (DS Pharma Biomedical, Osaka, Japan). All assays were replicated three times.