Hepatocyte cellular cytotoxicity was measured by determining the catalytic activity of released LDH using a Cytotoxicity Assay Kit (ThermoFisher Scientific). Briefly, hepatocytes isolated from WT and HEPiPLA2γKO mouse liver as described above were allowed to attach to 12-well plates (0.15 × 106 cells/well) for 2 days and then incubated in serum-free and phenol red-free Williams' E medium for 3 h. The attached cells were then exposed to DMSO vehicle alone (0.1%, v/v) or 5 μM A23187 for the indicated times. After stimulation with calcium ionophore, the cell media was collected, briefly centrifuged, and the resultant supernatant was collected for assay of LDH activity as described by the manufacturer's instructions. Maximum LDH release from the hepatocytes was determined by incubation of the attached cells with media containing 0.5% Triton X-100 for 1 min followed by measurement of LDH activity in the media.
Cytotoxicity assay kit
Manufactured by Thermo Fisher Scientific
Sourced in China, India
The Cytotoxicity Assay Kit is a laboratory instrument designed to measure the cytotoxic effects of various substances on cells. It provides a quantitative assessment of cell viability and proliferation.
Automatically generated - may contain errors
3 protocols using cytotoxicity assay kit
1
Hepatocyte Cytotoxicity Measurement by LDH Assay
2
Proanthocyanidins Modulate Cellular Processes
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Proanthocyanidins were bought from Nanjing Zelang Medical Technology Co. Ltd. (Nanjing, China), with a purity of over 98% as testified by high-performance liquid chromatography. SDS-proanthocyanidins GE Gel Preparation kit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Nanjing KeyGen Biology China (Nanjing, China), monodansylcadaverine (MDC) autophagy detection kit, Annexin V-APC/7-AAD apoptosis detection kit, First Strand cDNA Synthesis kit and Taq DNA polymerase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cytotoxicity assay kit, 3-methyladenine (3-MA), Hoechst 33342/propidium iodide (PI) double staining kit and MTT cell proliferation were purchased from Thermo Fisher Scientific (Shanghai, China).
3
LDH Cytotoxicity Assay Protocol
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LDH is a soluble cytosolic enzyme exist in almost all eukaryotic cells. The enzymes released into culture medium upon cell demise due to damage to the plasma membrane. The increase of the LDH activity in culture supernatant is correlated to the number of lysed cells. LDH Cytotoxicity Assay gives a colorimetric strategy to quantify LDH activity utilizing a mixed reaction containing lactate, NAD+, diaphorase, and INT. LDH catalyzes the diminishment of NAD+ to NADH within the presence of L-lactate, while the formation of NADH can be estimated in a coupled reaction in which the tetrazolium salt INT is decreased to a red formazan product.
The quantity of the very hued and soluble formazan can be estimated at 490 nm spectrophotometrically. Upon completion of the incubation, 50 μL of the upper phase was harvested from each well. The cells were then lysed with a cell lysis solution for 40 minutes at room temperature and the lysate was collected. LDH activity was estimated utilizing LDH release quantification (Fischer scientific, India) Cytotoxicity Assay Kit, as per producer’s guideline. At that point the level of LDH released surrounding environment was determined. In order to provide a positive one, a Cyclophosphamide reagent (5μg/mL) and the extract was added to the untreated cells to explore the cytotoxicity effect.
The quantity of the very hued and soluble formazan can be estimated at 490 nm spectrophotometrically. Upon completion of the incubation, 50 μL of the upper phase was harvested from each well. The cells were then lysed with a cell lysis solution for 40 minutes at room temperature and the lysate was collected. LDH activity was estimated utilizing LDH release quantification (Fischer scientific, India) Cytotoxicity Assay Kit, as per producer’s guideline. At that point the level of LDH released surrounding environment was determined. In order to provide a positive one, a Cyclophosphamide reagent (5μg/mL) and the extract was added to the untreated cells to explore the cytotoxicity effect.
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