Non radioactive cytotoxicity assay kit

Cytotoxicity was assessed by measuring the activity of LDH in the treatment medium using a nonradioactive cytotoxicity assay kit (Promega), as described by Kihara et al. [14 (link)]. PC12 and Caco-2 cells (1 × 10⁵ cells/flask) were cultured in medium with or without As³⁺ (5 μM), Cd²⁺ (5 μM), Pb²⁺ (5 μM), ALA (250 μM), and DHLA (50 μM) for 48 h. After 48 h of incubation, 50 µL of the medium was transferred to a 96-well plate, and then 50 µL of the substrate mixture containing tetrazolium salts was added to each of the sample wells in a 96-well plate. After a 30 min incubation at room temperature (25°C), 50 µL of the stop solution was added, and the amount of formazan dye formed was determined by measuring the absorbance at 490 nm using a microplate reader (Bio-Rad, CA, USA). LDH activity was expressed as LDH activity/1 × 10⁶ cells. This experiment was carried out in triplicate to ensure reproducibility.

CDC was assayed on MCF-7 cells that highly expressed the KH-1 antigen with a non-radioactive cytotoxicity assay kit (Promega). MCF-7 cells (25 μL, 1 × 10⁴ cells/well) were seeded into 96-well plates (Corning). Diluted sera (from preimmunization and 14 days after the fourth vaccination) were added and incubated at 37 °C for 2 h (the final concentration of sera was 1:20). After washing the cells twice, a rabbit complement (1:20) was added to the cells and incubated at 37 °C for another 4 h. After centrifugation, the cell supernatants (50 μL/well) were isolated and transferred to another 96-well plate. LDH assay reagents (50 μL/well) were added and incubated at room temperature protected from light for 30 min, and then a stop solution (1 M H₂SO₄, 50 µL/well) was added to each well of the plate. The absorptions of these plates were read at 490 nm wavelength using a microplate reader. The assays were performed in triplicate. The percentage of cell lysis was calculated according to the following formula: $$Celllysis(\%)=\frac{Experimental-TargetSpontaneous}{TargetMaxium-TargetSpontaneous}\times 100\%$$