Cytotox 96 non radioactive cytotoxicity assay ldh

Before the functional analysis, the transduction rates of NY-ESO-1 TCR and miR-H18 NY-ESO-1 TCR-transduced CD8+ T cells were adjusted with non-transduced cells from the same donor.
For the IFN-γ secretion assay, 5 × 10⁴ T2 cells per well were co-cultured with 5 × 10⁴ TCRβ+ NY-ESO-1 TCR, miR-H18 NY-ESO-1 TCR, or non-transduced CD8+ T cells in 200 μL/well TCM and in the presence of the indicated concentrations of cognate NY-ESO-1_157-165 (SLLMWITQC acid) or control MAGE-A1 (KVLEYVIKV acid) peptides (Discovery Peptides, Billingham, UK) for 18 h in U-bottom 96-well plates (TPP, Trasadingen, Switzerland). All samples were performed in duplicates. IFN-γ was quantified in the supernatants by ELISA.
For the CytoTox 96 non-radioactive LDH cytotoxicity assay (Promega), 1 × 10⁴ T2 cells per well were co-cultured with TCRβ+ NY-ESO-1 TCR, miR-H18 NY-ESO-1 TCR, or non-transduced CD8+ T cells in 100 μL/well LDH medium (RPMI-1640 phenol red [−], 5% FCS, 1% Pen/Strep, 1% NEAA, and 1% Na-Pyr) at the indicated E:T ratios and in the presence of the indicated concentrations of NY-ESO-1 or MAGE-A1 peptides in U-bottom 96-well plates. The plates were centrifuged at 250 × g for 4 min and incubated at 37°C for 4 h. All samples were performed in triplicates. LDH was quantified in the supernatants according to the manufacturer’s instructions.

The cytotoxicity of nanocomposites and rotating magnetic field were determined using LDH CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). Firstly, after the experimental treatment, the plates were centrifuged at 240× g for 4 min and the supernatant was transferred into new 96-well plates. Then, the transferred supernatant was mixed with Substrate Mix (Promega, Madison, WI, USA) and the plates were incubated for 30 min at room temperature (light-protected). Finally, Stop Solution (Promega, Madison, WI, USA) was added to the plates and the absorbance at 490 nm was measured using a spectrophotometer reader (Sunrise, Tecan, Männedorf, Switzerland) equipped with the Magellan Standard Software version 7.2 (Sunrise, Tecan, Männedorf, Switzerland). The nanocomposites—LDH assay components interaction was determined using different concentrations of the nanomaterials incomplete DMEM medium in the absence of cells [21 (link),61 ].

The cytotoxicity of G/C/GP hydrogel on iPSCs was examined using an extraction method^{15 (link),16 (link)}. A total of 0.1 g G/C/GP hydrogel was immersed in 1 ml mESCM in a 48-well culture plate at 37 °C. The supernatant from each well was collected on day 2 for the cytotoxicity test. iPSCs were seeded in 96-well cell culture plates at a density of 5000 cells per well and cultured in mESCM for 48 h. Cells were then cultured in the extraction medium obtained from the developed hydrogel. Crystal violet (HT90132 - crystal violet solution, Sigma-Aldrich) and lactate dehydrogenase (LDH, cytotox96 non-radioactive cytotoxicity assay, Promega, USA) were used to evaluate the cell viability and cytotoxicity of the developed hydrogel on iPSCs at 24 hours and 48 hours. The OD value of crystal violet and the LDH assay were measured at 570 and 490 nm, respectively, with an ELISA reader.

Cytocompatibility of the h-BN_AuNP nanocomposite was evaluated using the LDH CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). The LDH CytoTox 96^® Non-Radioactive Cytotoxicity Assay measures lactate dehydrogenase released due to cellular membrane damage. The amount of formazan converted from tetrazolium salt is proportional to the number of lysed cells. The LDH assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA) and the absorbance was measured at 490 nm using a microplate spectrophotometer (Absorbance Reader, Tecan, Männedorf, Switzerland). The interaction between the solution with the nanocomposite in the cell culture medium and LDH assay components was tested in the absence of cells. The percentage of LDH released after 24, 48 and 72-h exposure was calculated using the Formula (3): $\%\mathrm{LDH}\mathrm{released}=\frac{A490\mathrm{nm}\mathrm{of}\mathrm{treated}\mathrm{and}\mathrm{untreated}\mathrm{cells}-A490\mathrm{nm}\mathrm{of}\mathrm{control}}{A490\mathrm{nm}\mathrm{of}\mathrm{maximum}\mathrm{of}\mathrm{untreated}\mathrm{cells}-A490\mathrm{nm}\mathrm{of}\mathrm{control}}\times 100$
where A is absorbance.

At differentiation day 30, 100,000 cells were seeded onto 96-well plates (solid black plate; Corning). Two days later, cells were treated for 1 h with mitomycin C to eliminate remaining proliferative cells. Cells were fed on these plates until day 65, at which indicated treatments were added to the cells for 24–72 h. Assays were performed according to the manufacturer’s indications. CellTiter-Glo Luminescent Cell Viability Assay (Promega) which generates a luminescent signal proportional to the amount of ATP present in lysed cells, and LDH CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega, which generates a color proportional to the amount of lactate dehydrogenase (LDH) in lysed cells were used. All experiments were repeated at least three times and read for luminescence (CellTiter-Glo) or at 490 nm absorbance (CytoTox). Vehicle treated live cells were considered control, and percent control was calculated for CellTiter-Glo. For CytoTox, the ratio of LDH signal in the lysed cell divided by total LDH (released + lysed) was calculated. DAPI staining was used to visualize and quantify live cell nuclei morphology as another proxy for cell viability.