Ldh cytotoxicity colorimetric assay kit 2

GFP-rMSCs were cultured and hydrogen peroxide treated in 96-well plates. The culture media from the wells were used in an LDH assay to measure cytotoxicity caused by hydrogen peroxide in each well. The assay was performed in triplicate using an LDH-Cytotoxicity Colorimetric Assay Kit II (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer’s instructions with some minor modifications. The plates were centrifuged at 1500 rpm, and a medium was used in the assay. Higher levels of LDH indicated more cell death in the wells. The values were normalized to those of wells with media and no cells.

The cytotoxicity of A. squamosa extracts on Lovo and HCT-116 cell lines was quantitatively assessed through the measurement of lactate dehydrogenase (LDH). The cells were cultured for 24 h in 6-well plates (4 × 105 cells/well). After overnight growth, the culture medium was removed and replaced by 1 mL of culture medium. Then, the cells were treated with various concentrations of the leaves extract (1 μg/mL, 10 μg/mL, and 100 μg/mL) for 24 h. To estimate LDH activity, 10 μL of the culture supernatant was transferred to a new 96-well plate and the enzyme reaction was conducted in accordance with the manufacturer’s instructions (LDH cytotoxicity colorimetric assay kit II, BioVision, USA). The cytotoxicity was calculated as a percentage by using the following formula:
Cytotoxicity (%) = (test sample – negative control) / (positive control – negative control) × 100.

The cell leakage rate was determined by LDH Cytotoxicity Colorimetric Assay Kit II (BioVision Inc., Milpitas, CA, USA). HepG2 cells were incubated at a density of 5 × 10⁴ cells/mL in a 24-well plate for 24 h. The new culture media (1 mL) containing test samples, as described above, were added to each of the 24 wells, and the cells were incubated for 72 h. At the end of the incubation, 10 μL of media from each of the 24 wells was transferred to a 96-well plate, and 100 μL of LDH reagent was added and incubated for 30 min in dark at room temperature. The absorbance was read at 450 nm using ELISA reader (Molecular Devices). The LDH leakage was estimated from the ratio between the LDH activity in the medium and that of the whole cell content [12 (link)].

Cell viability was determined by an LDH-Cytotoxicity Colorimetric Assay Kit II (BioVision) according to manufacturer’s instructions. In brief, differentiated THP-1 cells were incubated with 100 or 200 μg/mL Aβ peptide for 48 h (see above). To obtain 100% LDH release the cells were incubated with 10 μL cell lysis solution at 37 °C 30 min prior to the measurement. 10 μL of the cell culture supernatant or 11 μL of the supernatant from lysed cells were transferred into a new clear 96-well plate (Greiner) and incubated with 100 μL LDH reaction mix for 30 min in the dark at room temperature and 80 rpm on a horizontal platform shaker (Heidolph). The absorbance of all samples was measured with a 96-well plate reader FluoStar Omega (BMG Labtech) at 450 nm and 650 nm. For data analysis, the absorbance at 650 nm was substracted from the absorbance at 450 nm.

Cytotoxicity was assessed by measuring the release of LDH into the media (LDH-Cytotoxicity Colorimetric Assay Kit II; BioVision) according to the manufacturer’s protocol.

LDH activity assays were performed as described previously [26 (link)]. Briefly, the supernatants from the cultures of ALS-As-iMNs and wild-type MNs were collected after a 4-week culture. Then, the supernatants were centrifuged, and the proteins in the supernatants were concentrated with protein concentrators (Thermo Scientific™). The LDH activity was measured in the concentrated supernatants with the LDH-cytotoxicity colorimetric assay kit II (BioVision). The genomic DNA (gDNA) in ALS-As-iMNs and wild-type MNs was extracted using the genomic DNA purification kit (Thermo Scientific™). The LDH activity values were normalized to the amount of gDNA of the ALS-As-iMNs and wild-type MNs.