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Lactate dehydrogenase assay

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The Lactate dehydrogenase (LDH) assay is a colorimetric enzymatic assay used to measure the activity of the LDH enzyme. LDH catalyzes the conversion of lactate to pyruvate, with the concurrent reduction of NAD+ to NADH. The rate of NADH formation, which is proportional to the LDH activity, is measured spectrophotometrically.

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Lab products found in correlation

96 well plate, by Greiner (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Hydroxychloroquine sulfate, by Merck Group (1 mentions) Bafilomycin, by Enzo Life Sciences (1 mentions) Imipramine hydrochloride, by Merck Group (1 mentions) Elisa kit, by R&D Systems (1 mentions)

6 protocols using lactate dehydrogenase assay

1

Cell Viability Assay for HPAC Cells

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HPAC cell lines were seeded in 96-well plates (Greiner) and inoculated at the indicated MOIs. After 1 hour, cells were washed once with PBS and fresh infection media was added. A lactate dehydrogenase assay (Promega, The Netherlands) was used to determine cell viability as described before [31 (link)].
de Graaf J.F., van Nieuwkoop S., Bestebroer T., Groeneveld D., van Eijck C.H., Fouchier R.A, & van den Hoogen B.G. (2022). Optimizing environmental safety and cell-killing potential of oncolytic Newcastle Disease virus with modifications of the V, F and HN genes. PLoS ONE, 17(2), e0263707.
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2

Neurotoxicity Experiments with Microglia and Neurons

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BV2 immortalized microglia and SH-SY5Y neuroblastoma were used for neurotoxicity experiments. BV2 microglia were plated at 50,000 cells per square centimeter, and SH-SY5Y were plated separately at 100,000 cells per square centimeter. Microglia were treated with SR-targeting antibodies or antioxidant loaded nanoparticles, or non-SR targeting and no-antioxidant loaded NPs for 24 hours, followed by co-treatment with either 1µg/mL LPS (Sigma), 20 µM monomeric ASYN, which has been demonstrated to activate BV2 cells 37 (link) or the same mass of fibrillar ASYN. SH-SY5Y cells were treated with media conditioned by the ASYN- or LPS-stimulated microglial cells for 24 hours. Cytotoxicity in response to stimulated microglia conditioned media was quantified in SH-SY5Y populations was quantified using a lactate dehydrogenase assay (Promega), normalized to a positive control of totally lysed SH-SY5Y cells, and to untreated BV2-conditioned SH-SY5Y cells.
Bennett N.K., Chmielowski R., Abdelhamid D.S., Faig J.J., Francis N., Baum J., Pang Z.P., Uhrich K.E, & Moghe P.V. (2016). Polymer Brain-Nanotherapeutics for Multipronged Inhibition of Microglial α-Synuclein Aggregation, Activation, and Neurotoxicity. Biomaterials, 111, 179-189.
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3

Resveratrol and ONX-0914 Effects on Monocytes

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Monocytes (2×104 cells/well) were cultured in a 96 well plate for 24 h and then incubated with different concentrations of resveratrol and ONX-0914. Stock solutions of resveratrol and ONX-0914 were prepared in dimethyl sulfoxide (DMSO) followed by dilution in RPMI1640 complete media containing 0.2 % DMSO. After drug treatment, cells were first washed with 100 μl/well PBS and then, 100 μL MTT (1 mg/ml) stock solutions was added to each well. Cells were incubated for 2 h in the dark, washed and the reaction was terminated by addition of 100 μL of isopropanol. Plate was allowed to shake for 10 min at room temperature and absorbance was read at 570 nm using plate reader Cytation 3. We also performed lactate dehydrogenase assay (Promega) and observed similar results.
Silswal N., Reddy N.S., Qureshi A.A, & Qureshi N. (2018). RESVERATROL DOWNREGULATES BIOMARKERS OF SEPSIS VIA INHIBITION OF PROTEASOME’S PROTEASES. Shock (Augusta, Ga.), 50(5), 579-588.
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4

Glutamate-Induced Cell Death Assay

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In order to measure cell death as a response to the stimulation with glutamate and NMDA, we used a lactate dehydrogenase assay from Promega (code: G1780). At each time point supernatant was collected and stored in −80 °C until all time-points were collected. For the analysis 50 μl of the sample was pipetted into a 96-well plate and mixed with 50 μl reconstituted substrate mix. Then incubation for 30 min in a light protected condition followed before 50 μl of stop solution was added. Finally the absorbance was read at 490 nm.
Spang C., Backman L.J., Le Roux S., Chen J, & Danielson P. (2017). Glutamate signaling through the NMDA receptor reduces the expression of scleraxis in plantaris tendon derived cells. BMC Musculoskeletal Disorders, 18, 218.
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5

Lactate Dehydrogenase Assay Protocol

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The lactate dehydrogenase assay (Promega, Madison, WI, USA) was performed according to the manufacturer’s protocol.
De Luna-Preitschopf A., Zwickl H., Nehrer S., Hengstschläger M, & Mikula M. (2017). Rapamycin Maintains the Chondrocytic Phenotype and Interferes with Inflammatory Cytokine Induced Processes. International Journal of Molecular Sciences, 18(7), 1494.
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6

Inflammasome Activation by Crystalline Silica

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BMDM were platted in a flat-bottom, tissue culture- treated 96-well plates at 1×105 cells/well in 100 μL of RPMI complete media and incubated with lipopolysaccharide (LPS) (20 ng/mL) for inflammasome priming. Cells were treated with 25 μM of hydroxychloroquine sulfate (Sigma-Aldrich cat. H0915-5MG), 25 μM imipramine hydrochloride (Sigma-Aldrich cat. 10899-5G), or 100 nM bafilomycin (EnzoLife Sciences cat. BML-CM110-0100) for 30 minutes prior to addition of cSiO2. Cells were exposed to various doses (0-50 μg/mL) of cSiO2 and plates were incubated in a 37°C water-jacketed CO2 incubator (ThermoForma, Houston, TX) for 24 hours. Toxicity induced by cSiO2 was determined by two complementary assays, a lactate dehydrogenase assay (Promega, cat. G1780) and a common colorimetric tetrazolium viability (MTS) assay (Promega, cat. G3580), and read on a plate reader (Molecular Devices SpectraMax M4 colorimetric microplate reader). In order to avoid artifacts in the optical density values, the MTS reaction was transferred to a clean plate to separate it from the cell/particle mixture adhered to the plate bottom. Data were normalized to a percent relative to the no particle, no HCQ control cells. NLRP3 inflammasome activation was assayed by measuring the release of IL-1β by using a commercially available ELISA kit (R&D Systems, cat. DY201).
Burmeister R., Rhoderick J.F, & Holian A. (2019). Prevention of Crystalline Silica-Induced Inflammation by the Anti-malarial Hydroxychloroquine. Inhalation toxicology, 31(7), 274-284.
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