Pierce ldh cytotoxicity assay kit

The cytotoxicity of lin-SB056-1 and (lin-SB056-1)₂-K towards A549 and THP-1 cells was evaluated to exclude a possible correlation between reduction in cytokine levels and decrease in cell numbers. To this end, culture supernatants of cells stimulated with LPS in the presence of the peptides were employed to quantify the amount of cytoplasmic LDH released by dead cells. Cells incubated in culture medium alone or in the presence of LPS served as an indication of spontaneous cell death (cell viability controls), while cells treated with a commercial lysis buffer (Pierce LDH cytotoxicity assay kit, Thermo Fisher Scientific) were used as cell death control (100% cell lysis). The enzymatic activity of LDH in cell supernatants was measured using the Pierce LDH cytotoxicity assay kit (Thermo Fisher Scientific) following manufacturer’s instructions. The cytotoxic effect was determined as follows: cytotoxicity (%) = [(LDH activity of A/LDH activity of B) × 100], where A represents peptide-treated or untreated cells (both in the presence and absence of LPS), and B corresponds to the cell lysis control.

Cells were seeded in 96-well plates and cultured in an atmosphere containing 5% CO₂, at 37 °C. Six hours after cell seeding, the medium was replaced with the similar medium containing 1, 10, or 100 capsules per cell. Triton X-100 was used as a positive control. Within 72 h after the addition of the CeGdO_2−x NP-loaded capsules, the level of lactate dehydrogenase in the culture medium was determined, according to the manufacturer’s protocol (The Thermo Scientific™ Pierce™ LDH Cytotoxicity Assay Kit, Cambridge, UK). Absorbance of the solution was measured at wavelengths of λ = 490 nm and λ = 640 nm, using the Microplate Reader ThermoMultiskan Ascent 96 & 384 (Thermo Fisher Scientific, Cambridge, UK).

DIV 14 cortical neurons were subjected to water control or glutamate insult (200 μM glutamate for 5 min). Prior to the insult, half of the media was removed (and saved for later use as conditioned media) from each well and replenished with fresh feeding media. After the insult, media was exchanged with a 1:1 mix of fresh and conditioned media, and neurons were returned to 37 °C and 5% CO2 for 20 to 24 h. Media samples to be tested for cell death were collected 1 h before or 24 h after glutamate insult, as indicated. Cell death was then assessed as described previously (23 (link), 31 ) by measuring the lactate dehydrogenase (LDH) released from the cells into the media using a Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific).