Ldh assay kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The LDH assay kit is a laboratory tool used to measure the activity of the enzyme lactate dehydrogenase (LDH) in biological samples. LDH is an important marker for various cellular processes and can be used to assess cell health and damage. The kit provides the necessary reagents and protocols to quantify LDH levels colorimetrically or fluorometrically.

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Close spelling variants of this product

LDH assay (22 citations) LDH kit (3 citations)

49 protocols using ldh assay kit

Compound-Mediated Cytotoxicity Assessment

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In order to test for compound-mediated cytotoxicity, both cell types were separately incubated in 96-well flat-bottomed cell culture plates (BD Biosciences, Heidelberg, Germany) together with the various compounds used in the study for up to 48 h. To analyze for cell-mediated cytotoxicity the cells were co-cultured in a 96-well HTS transwell tissue culture system (Corning, Acton, Massachusetts, USA) for up to 48 h. Potential cytotoxic effects were determined by measuring lactate dehydrogenase (LDH) activity in cell culture media using a commercially available LDH assay kit (Pierce Biotechnology, Rockford, Illinois, USA) according to the manufacturer’s specifications.
Compound-affected cell viability was reduced by high levels of LPS and UHMWPE particles as compared to the corresponding negative control at selected time points. However, no remarkable changes were observed with overall viability ranging between 95–100 % for MG-63 and 94–99 % for THP-1 cells in comparison to the untreated control (93–100 % and 98–100 %, respectively). Also, viability was not considerably decreased in co-culture conditions ranging between 97–100 %.

Jablonski H., Rekasi H, & Jäger M. (2016). The influence of calcitonin gene-related peptide on markers of bone metabolism in MG-63 osteoblast-like cells co-cultured with THP-1 macrophage-like cells under virtually osteolytic conditions. BMC Musculoskeletal Disorders, 17, 199.

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Quantifying Cell Viability via LDH Assay

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To determine cell viability, the lactate dehydrogenase (LDH) assay kit (88954) from Pierce Biotechnology was used. The remaining cell viability was determined by lysing cells in 50 μl 1× lysis buffer, and 50 μl LDH substrate was added and incubated until a sufficient colorimetric change was visible. The reaction was stopped using the LDH stop solution, and the plate was read at 490 and 680 nm for background measurement, which were subtracted. Untreated sample was set at 100% viability, and the proportion of remaining cell viability was determined for all other conditions.

Gram A.M., Wright J.A., Pickering R.J., Lam N.L., Booty L.M., Webster S.J, & Bryant C.E. (2020). Salmonella Flagellin Activates NAIP/NLRC4 and Canonical NLRP3 Inflammasomes in Human Macrophages. The Journal of Immunology Author Choice, 206(3), 631-640.

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Cerebral Cortex Tissue LDH Assay

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Lysates from cerebral cortex tissues after BDL were added to a 96-well plate to measure lactate dehydrogenase (LDH) levels using an LDH assay kit from Invitrogen. The reaction mixture in the assay kit was added to each well. After 30 min of incubation, the stop solution was added to each well, and absorbance (at 490/680 nm) was measured by using a microplate reader (Molecular Devices).

Cheon S.Y., Jo D., Kim Y.K, & Song J. (2022). Long Noncoding RNAs Regulate Hyperammonemia-Induced Neuronal Damage in Hepatic Encephalopathy. Oxidative Medicine and Cellular Longevity, 2022, 7628522.

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LDH Cytotoxicity Assay Protocol

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Cell conditioned media were transferred to a multi-well plate to detect released LDH levels due to cell membrane damage and cytotoxicity using an LDH assay kit (Invitrogen). The reaction mixture was added to each sample well, mixed well by tapping, and incubated at RT for 30 minutes. The stop solution was then added to each well, and the plates were read at 490/680 nm using a VERSA max microplate reader (Molecular Devices).

Lee S.K., Kam E.H, & Cheon S.Y. (2023). Autophagy Enhancers Regulate Cholesterol-Induced Cytokine Secretion and Cytotoxicity in Macrophages. Journal of Lipid and Atherosclerosis, 12(2), 189-200.

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Evaluating AGE-Induced Cytotoxicity with LDH Assay

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To determine the cytotoxicity of the AGEs, we performed lactate dehydrogenase (LDH) assay. LDH assay kit was obtained from Invitrogen. LDH production was determined as described previously [31 (link)]. Briefly, BV2 cells were treated with different concentrations of MGO-AGE and were incubated for 24 h. Conditioned medium (50 μL) from the treated cells were collected and mixed with equal amounts of LDH mixture (LDH substrate and buffer). This resulted in a color change into a brown-red color because of the conversion of lactate to pyruvate in the presence of LDH enzyme, which was evaluated my measuring the absorbance at 450 nm. This experiment was performed exactly as described by LDH assay kit protocol from Thermo Fisher Scientific/Invitrogen (catalogue no. C20300, Waltham, MA, USA).

Subedi L., Lee J.H., Gaire B.P, & Kim S.Y. (2020). Sulforaphane Inhibits MGO-AGE-Mediated Neuroinflammation by Suppressing NF-κB, MAPK, and AGE–RAGE Signaling Pathways in Microglial Cells. Antioxidants, 9(9), 792.

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Cytotoxicity Evaluation of Benzothiazine Derivatives

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The toxicity assay assessed benzothiazine derivatives' possible toxicity to the host cells. The assay was performed as per the previous protocol [27 ]. The toxicity of compounds was determined against human keratinocyte (HaCaT) cells using a lactate dehydrogenase (LDH) assay kit. Briefly, a 96-well plate was loaded with cells and incubated at 37 ^°C for 24 h to form a monolayer. The monolayer was then challenged with 50 and 100 μM concentrations of benzothiazines in 200 μL of RPMI-1640. The cells were kept in a standard CO₂ incubator at 37 ^°C for 24 h. Untreated cells in RPMI-1640 alone were used as a negative control, while Triton X-100 was treated as a positive control. After incubation, the quantity of LDH released by injured or dead cells was used to evaluate the degree of cell death. The extent of LDH was enumerated using an LDH assay kit (Invitrogen, Illinois, USA). The reading was recorded using a microplate reader at 490 nm. The formula ((Sample absorbance - negative control absorbance)/(Positive control absorbance - negative control absorbance) × 100) was used to determine the percentage of chemical toxicity.

A.l.i.s.h.b.a., Ahmed U., Taha M., Khan N.A., Salar U., Khan K.M., Anwar A, & Siddiqui R. (2023). Potential anti-amoebic effects of synthetic 1,4-benzothiazine derivatives against Acanthamoeba castellanii. Heliyon, 10(1), e23258.

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Hippocampal LDH Cytotoxicity Assay

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Lysed hippocampal tissues or microglial supernatants were added to a 96-well plate to detect the LDH levels. An LDH assay kit was purchased from Invitrogen (Carlsbad, CA, USA). The reaction mixture was prepared according to the manufacturer's instructions and added to each well of the plate. After incubation, the stop solution was added, and absorbance values at 490/680 nm were obtained using a microplate reader (Molecular Devices).

Cheon S.Y., Kim M.Y., Kim J., Kim E.J., Kam E.H., Cho I., Koo B.N, & Kim S.Y. (2023). Hyperammonemia induces microglial NLRP3 inflammasome activation via mitochondrial oxidative stress in hepatic encephalopathy. Biomedical Journal, 46(5), 100593.

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Cytotoxicity Assessment of BMDM, hMDM, and iPSDM

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BMDM were seeded at 200 000 cells per well in 12-well plates, treated as indicated, and cytotoxicity assayed by LDH release assay the following day. At the end of the incubations, cell culture supernatants were collected, cleared of debris by centrifugation for 5 min at 500 x g. The cells were washed once with PBS then lysed in lysis buffer provided in the LDH assay kit (Invitrogen C20300). Supernatants and lysates were assayed for LDH using an LDH colorimetric assay kit as per the manufacturer's instructions (Invitrogen C20300). hMDMs were seeded at 500 000 cells per well in 12-well plates and differentiated as described above. iPSDMs were seeded at 180 000-450 000 cells per well in 12-well plates and differentiated as described above. After cells were stimulated as described in figure legends, supernatants were harvested and LDH release was assayed (Invitrogen C20301). Briefly, equal amounts of supernatant and reaction mixture was mixed and incubated for 30 minutes, followed by absorbance reads at 490 and 655 nm. To calculate the LDH release, the 655 nm background was subtracted from the 490 nm absorbance reads before further processing.

Borges J.P., Sætra R.S., Volchuk A., Bugge M., Kilburn B.R., Goldenberg N.M., Flo T.H., & Steinberg B.E. (2021). Glycine targets NINJ1-mediated plasma membrane rupture to provide cytoprotection.

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Quantifying Bacterial LDH Activity

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LDH catalyses the conversion of lactate to pyruvate via reduction of NADP to NADPH, which is associated with the formation of tetrazolium giving the red colour. The release of LDH was determined using the LDH assay kit (Fisher Scientific/ Cat No. 88954). Briefly, bacterial cells were cultured in 96-well plates (10⁴ cfu/ml) in the Muller-Hinton media in triplicate at 37°C overnight. The cells were then treated with 10 μl laser Ag NPs or 10 μl dH₂O as control in a final concentrations of NPs to be 2–10 μg/ml and further incubated at 37°C overnight. Ten μl of lysis buffer was then added and mixed by gently tapping and incubated at 37°C for 45 minutes. Fifty μl cell lysate from each sample were then transferred to a new 96-well plate and 50 μl reaction buffer was added to each well and mixed gently. The plate was then left at room temperature for 30 minutes in dark before 50 μl stop solution was added to each well and mixed by gentle tapping. Finally, optical density (OD, Absorbance) was measured at 490 nm using a spectrophotometer (Omega).

Korshed P., Li L., Liu Z, & Wang T. (2016). The Molecular Mechanisms of the Antibacterial Effect of Picosecond Laser Generated Silver Nanoparticles and Their Toxicity to Human Cells. PLoS ONE, 11(8), e0160078.

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Assessing Antimicrobial Activity of Laser Ag-TiO2 NPs

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E. coli at 10⁴ cfu/mL were cultured in 96-well plates in 200 µL Muller–Hinton media in triplicate at 37°C overnight. The cells were then treated with 10 µL laser Ag-TiO₂ NP concentrations (10, 20 and 50 µg/mL) or 10 µL dH₂O as control and further incubated at 37°C overnight. Lysis buffer 10 µL from the LDH assay kit (Fisher Scientific, Loughborough, UK/Cat No 88954) was then added and mixed by gently tapping and incubated at 37°C for 45 minutes. Cell lysate 50 µL from each sample was then transferred to a new 96-well plate and 50 µL reaction buffer from the kit was added to each well and mixed gently. The plate was then left at room temperature for 30 minutes in dark before 50 µL stop solution was added to each well and mixed by gentle tapping. Finally, OD absorbance was measured at 490 nm using the spectrophotometer (Omega).

Korshed P., Li L., Liu Z., Mironov A, & Wang T. (2017). Antibacterial mechanisms of a novel type picosecond laser-generated silver-titanium nanoparticles and their toxicity to human cells. International Journal of Nanomedicine, 13, 89-101.

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