The cytotoxicity of the focA mutants was evaluated by LDH release from Salmonella-infected cells. Cell culture and bacterial infection were performed as in the mutant screen described above. DMEM with 10 μg/mL gentamicin was added for 3 h, and the LDH level released in the cell medium was detected by an LDH cytotoxicity assay detection kit (Beyotime, Nantong, China).
Ldh cytotoxicity assay detection kit
Manufactured by Beyotime
Sourced in China
The LDH cytotoxicity assay detection kit is a colorimetric assay designed to measure the release of lactate dehydrogenase (LDH) from damaged or dying cells. LDH is a stable cytoplasmic enzyme that is released upon cell lysis or membrane damage, and its activity can be quantified using a coupled enzymatic reaction that results in a colored product. This kit provides a simple and reliable method for assessing cell viability and cytotoxicity in various experimental settings.
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Lab products found in correlation
Spectramax 190 microplate reader, by Molecular Devices (1 mentions)
Celltiter lumi plus luminescent cell viability assay kit, by Beyotime (1 mentions)
Z vad fmk, by Beyotime (1 mentions)
Infinite f50 absorbance microplate reader, by Tecan (1 mentions)
Synergy 2 multi mode microplate reader, by Agilent Technologies (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
13 protocols using ldh cytotoxicity assay detection kit
1
Evaluating Cytotoxicity of focA Mutants
2
LDH Assay for UVB-Induced Cell Death
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The LDH assay was employed to evaluate cell death based on the idea that LDH would be released from the cytoplasm through the damaged plasma membrane when cell death occurred46 (link). As a result, quantification of the LDH level in cell culture supernatants was used to evaluate cell death in our study with an LDH cytotoxicity assay detection kit (Beyotime Biotechnology) according to the manufacturer’s instructions. Briefly, HaCaT cells were treated with or without 50 mJ/cm2 UVB radiation; then, the cells were cultured in the presence or absence of 100 mM trehalose for 12 hours. Cell culture supernatants were collected for measurement. The optical density was measured at a 490 nm wavelength. The percentage of cell death was calculated with the formula: Cytotoxicity level (percentage of cell death) = (OD of experimental release (trehalose treatment, UVB exposure, or trehalose treatment after UVB exposure, respectively) - OD of spontaneous release (cell with normal culture))/(OD of maximum release (cells treated with LDH releasing reagent provided in kit) - OD of spontaneous release (cell with normal culture)).
3
LDH-Based Cytotoxicity Quantification
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Cell death was evaluated by the quantification of plasma membrane damage which resulted in the release of lactate dehydrogenase (LDH). The level of LDH released in the cell culture supernatant was detected by LDH cytotoxicity assay detection kit (Beyotime, shanghai, China) following the manufacturer's instructions.
4
Quantifying Cytotoxicity via LDH Release
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Cell death was also evaluated via the quantification of plasma membrane damage that resulted in the release of lactate dehydrogenase (LDH). The amount of LDH released into the cell culture supernatant was detected by an LDH cytotoxicity assay detection kit (Beyotime, China) following the manufacturer's instructions.
5
Cytotoxicity Evaluation of PZQ Enantiomers
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To further confirm the cytotoxicity of compounds (R)-PZQ, (S)-PZQ, and rac-PZQ against the eight cell lines, cell death was evaluated by the quantification of plasma membrane damage that resulted in the release of lactate dehydrogenase (LDH). These compounds were dissolved in DMSO and diluted with culture medium. The cells cultured in the exponential growth phase were treated with different concentrations (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM) of PZQ enantiomers at a density of 5×104 cells/100 μL per well and incubated for 48 hours at 37°C in 5% CO2 incubator. Then, the level of LDH released in the cell culture supernatant was detected by LDH cytotoxicity assay detection kit (Beyotime Institute of Biotechnology, Jiangsu, People’s Republic of China) following the manufacturer’s instructions. The negative (cells and solvent) and positive (cells treated with rac-PZQ) controls were run in parallel. All assays were carried out in triplicate. The results were expressed in the cell viability rates under different drug concentrations on the four cell lines and are described in Figure 4 .
6
Quantifying Cell Cytotoxicity via LDH Assay
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The LDH assay was performed using the LDH cytotoxicity assay detection kit (Beyotime, China), according to the manufacturer’s instructions. The assay measures the conversion of a tetrazolium salt to a red formazan product, detectable by absorbance measurement at 490 nm. For this, a SpectraMax 190 Microplate Reader (Molecular Devices) was used.
7
Measuring Necrotic Cell Death via LDH
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LDH is a cytoplasmic enzyme in cells and releases rapidly after the plasma membrane is damaged. Thus, LDH is used as an indicator of necrotic cell death [40 (link)]. CD8+ T cells cocultured with Ishikawa and RL95-2 cells with shKIF2C and shNC at a 2 : 1 (CD8+ T cell : tumor cell) ratio; after a 3-day incubation, the cell culture supernatant was collected, and the level of LDH was detected by LDH cytotoxicity assay detection kit (Beyotime, Shanghai, China) according to manufacturer's protocol.
8
Cell Viability and Cytotoxicity Assays
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Cells were seeded onto 96-well plates at a density of 30,000 cells per well. The second day after seeding, the cells were counted and pretreated with 20 μM Z-VAD-FMK (Beyotime Biotechnology), 1 μM necrosulfonamide (MCE, Monmouth Junction, NJ, USA), 1 μM GSK’872 (MCE), 10 μM necrostatin-1 (MCE), or DMSO vehicle control for infection or transfection. LDH released was determined by the LDH cytotoxicity assay detection kit (Beyotime Biotechnology). The cell culture supernatant was collected for detection. The absorbance was read at 490 nm with Infinite® F50 Absorbance Microplate Reader (Tecan, Switzerland). Cell viability was measured based on the intracellular ATP levels using CellTiter-Lumi™ Plus Luminescent Cell Viability Assay Kit (Beyotime Biotechnology). An equal volume of Celltiter-LUMI™ Plus reagent was added to the cell culture medium to induce cell lysis by oscillation. After incubation at room temperature, the luminescence signal was measured by SynergyTM 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).
9
Evaluating bEnd.3 Cell Viability
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The viability of bEnd.3 cells was evaluated by quantifying plasma membrane damage, which resulted in the release of LDH. The level of Lactate dehydrogenase (LDH) released in the cell culture supernatant was detected using an LDH cytotoxicity assay detection kit (Beyotime, China) according to the manufacturer’s instructions.
10
Quantifying Cell Membrane Damage
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Cell death was evaluated by the quantification of plasma membrane damage which resulted in the release of lactate dehydrogenase (LDH). The level of LDH released in the cell culture supernatant was detected by LDH cytotoxicity assay detection kit (Beyotime, China) following the manufacturer's instructions.
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