Lactate dehydrogenase (In vitro toxicology LDH assay kit, Sigma-Aldrich) was measured in the conditioned media according to the manufacturer’s instructions [37 (link)].
In vitro toxicology ldh assay kit
Manufactured by Merck Group
Sourced in Germany, United States, Australia
The In vitro toxicology LDH assay kit is a laboratory product designed to measure the activity of the enzyme lactate dehydrogenase (LDH) in cell culture samples. LDH is released into the cell culture medium upon cell damage or death, and its quantification can serve as an indicator of cytotoxicity.
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4 protocols using in vitro toxicology ldh assay kit
1
Measuring Lactate Dehydrogenase Activity
2
Placental explant LDH and hCG assay
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Briefly, we assayed for lactate dehydrogenase (In vitro toxicology LDH assay kit, Sigma–Aldrich Chemie GmbH, Taufkirchen, Germany) and hCG release into the media after a 2 h incubation. After placental preparation the control was obtained by sonicating fresh placental villous explants (equivalent to the amount of explants used per well) from the same placentae in the same culture media used for experimentation. LDH values were quantified as a proportion of the 100% LDH control. Quantitative detection of hCG was performed with a solid-phase, two-step chemiluminescent immunometric assay (Immulite/Immulite 1000 analyzer, Siemens).
3
Cytotoxicity Assay for Caco2 and HepG2 Cells
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A cytotoxicity assay measuring lactate dehydrogenase (LDH) release was performed using human intestinal epithelial (Caco2) and human hepatocytic (HepG2) cells. The cells were cultivated in Eagle's minimum essential medium (MEM) containing 2 mM L-glutamine, 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 mg/mL streptomycin sulfate, 0.25 mg/mL amphotericin B, and 1% nonessential amino acids (NEAA; all PAA) at 37°C in a humidified atmosphere (95% relative humidity) containing 5% CO2.
The cells were seeded in 96-well plates (5 × 104 per well) and incubated until a confluent cell layer developed. For the cytotoxicity assay, cells were treated with 50 μL fungal fraction and 50 μL MEM for 24 h at 37°C and LDH leakage was determined using the LDH in vitro toxicology assay kit according to the manufactures' instructions (Sigma-Aldrich, USA). The percentage of dead cells was calculated using a standard curve of serial diluted lysed cells (100% dead cells). Each experiment was performed three times in triplicate.
The cells were seeded in 96-well plates (5 × 104 per well) and incubated until a confluent cell layer developed. For the cytotoxicity assay, cells were treated with 50 μL fungal fraction and 50 μL MEM for 24 h at 37°C and LDH leakage was determined using the LDH in vitro toxicology assay kit according to the manufactures' instructions (Sigma-Aldrich, USA). The percentage of dead cells was calculated using a standard curve of serial diluted lysed cells (100% dead cells). Each experiment was performed three times in triplicate.
4
Assessing Enoxaparin's Cytotoxicity Impact
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The effect of enoxaparin fractionon cell viability after 72 hours of incubation was assessed using two methods, trypan blue dye exclusion assay and lactate dehydrogenase (LDH) release into culture supernatant. Both assays employed routinely used methods. Cytotoxicity of treatment was determined using the LDH in-vitro toxicology assay kit (Sigma-Aldrich, NSW, Australia), according to the manufacturer’s instructions. Briefly, PBMC culture supernatants were centrifuged at 250g for 4 minutes. An aliquot containing 50 μL of cleared supernatant was mixed with 100 μL of a solution containing LDH assay substrate, LDH dye and LDH cofactor and incubated at room temperature for 20 minutes before the reaction was terminated by the addition of 15 μL of 1 N hydrochloric acid. Absorbance at 490 nm was measured spectrophotometrically using a plate reader (Spectra Max M2 microplate reader, Sunnyvale, CA). Each sample was measured in triplicate.
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