Extracellular lactate dehydrogenase (LDH) release following treatment of L929 cell line with 0.2 g/mL GHMS, GHS, 50 μM metformin, or no treatment was determined using a CytoTox 96 Assay Kit (Promega Corporation, Madison, WI, USA) to evaluate possible toxicity.
Cytotox 96 assay kit
Manufactured by Promega
Sourced in United States
The CytoTox 96 assay kit is a colorimetric assay designed to quantify the amount of lactate dehydrogenase (LDH) released from damaged cells. It provides a simple and reliable method for measuring cytotoxicity.
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Lab products found in correlation
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Zombie yellow fixable viability kit, by BioLegend (2 mentions)
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Mouse il 1β, by Thermo Fisher Scientific (2 mentions)
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Multiskan go plate reader, by Thermo Fisher Scientific (2 mentions)
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Ac ietd cho, by Merck Group (1 mentions)
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Il 1 beta mouse elisa kit, by Thermo Fisher Scientific (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Lsr fortessa instrument, by BD (1 mentions)
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Nkp46 29a1.4, by BioLegend (1 mentions)
65 protocols using cytotox 96 assay kit
1
Extracellular LDH Release in L929 Cells
2
Cytotoxicity Assessment of Clostridium perfringens
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Bone marrow-derived macrophages were seeded at a density of 0.35 × 106 cells per well in 24-well plates containing 10% FBS-RPMI 1640. The LPS (100 ng/ml, 2 h)-primed cells were infected with C. perfringens at a multiplicity of infection (MOI) of 2.5 (ATCC13124) or 25 (strain 13) per cell. The plates were incubated at 37°C. At the indicated times after infection, lactate dehydrogenase (LDH) activity in the culture supernatants was measured using a CytoTox 96 assay kit (Promega, Madison, WI, United States), in accordance with the manufacturer’s protocol. The following formula was used to calculate the amount of LDH released: [(OD490 sample release-OD490 negative control release)/(OD490 positive control release-OD490 negative control release)] ×100, where OD490 negative control release represents the amount of LDH released into the culture supernatant from uninfected cells and OD490 positive control release represents the amount of LDH released after lysis of the uninfected cells. Cytokines released in the culture supernatants were quantified by ELISA (eBioscience). The endpoint of the experiments was defined as 1 h post-infection (hpi), because C. perfringens are extracellular pathogens and multiply quickly in cell culture media.
3
LDH Release and Apoptosis Measurement in BMDMs
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For LDH release, BMDMs were plated into TC-treated plates in complete DMEM containing 10% L929 supernatant. LDH release was measured from cell supernatants of infected BMDMs and quantified using the Cytotox96 Assay kit (Promega) according to manufacturer’s instruction. Flow cytometry measurement of CC3 was performed by harvesting infected BMDMs with cold PBS containing 2% EDTA and staining with the Zombie Yellow Fixable Viability kit (BioLegend) before fixation and permeabilization (BD Cytofix/Cytoperm kit). Cells were then stained for CC3 (9661; Cell Signaling), followed by secondary anti–rabbit Alexa Fluor 488. Samples were run on an LSRFortessa and analyzed using FlowJo Tree Star software.
4
Cytotoxicity Evaluation of Peptide Nanoformulations
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The cell culture supernatants (50 μl) from the MDMs treated with Pep-H and its nanoformulations for 72 hrs were subjected to LDH assay to confirm the cell cytotoxicity. The assay was performed using Cytotox 96 assay kit (Promega) as per manufacturer’s instructions. For calculating %cytotoxicity by the peptide and its nanoformulations, a positive control of tritonX-100 leading to maximum LDH release was included. The absorbance obtained in the presence of test samples and positive control was used to calculate % cytotoxicity as A490 (Sample)/A490(Positive control) × 100.
5
Cytotoxicity Assay for Tricin and Flavopiridol
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To measure cytotoxic effects of tricin and flavopiridol, the CytoTox 96 assay kit (Promega Japan Inc.) was used according to the manufacturer's instructions. The CytoTox 96 assay kit quantitatively measures a cytosolic enzyme, lactate dehydrogenase (LDH), which is released upon cell lysis. A total of 10 000 cells were plated in a 96‐well tissue culture plate in triplicate, and serum‐free medium containing various concentrations of the drug was present before the cytotoxicity assays. The released LDH is able to convert the substrate tetrazolium salt into a red formazan product, which can be measured at 492 nm in a 96‐well tissue culture plate. To determine the percent cytotoxicity, the amount of LDH released by cells after drug treatment was compared and normalized with the amount of LDH released after complete cell lysis.
6
Cytotoxicity Assay for Membrane Damage
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The CytoTox96® Assay kit (Promega S.A. G4000, Whitehead Scientific, Johannesburg, South Africa) was used to measure the LDH released by treated and untreated cells. The enzyme LDH is present in the cytosol of live cells that is released in the culture media due to membrane damage forming a red formazan complex by reacting with the assay fluid, which was read at 490 nm. Hence, the measure of LDH diffused in media gives the measure of membrane damage or cytotoxicity. Briefly, equal volumes of (50 µL) substrate and spent media from each group were added, mixed, and incubated for 30 min in the dark at RT for the formation of formazan complex, which was read using a Multilabel Counter.
7
Quantifying Cytokine and LDH Release in BMDM Infection
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BMDMs were seeded at a density of 4 x 105 cells/well in 24-well plates containing RPMI 1640 supplemented with 10% FBS. At the times indicated after the infection, the cytokines released into the culture supernatants were quantified using ELISA kits. The following ELISA kits were purchased commercially; mouse IL-1β (Invitrogen, 88-7013-88), mouse IL-18 (Invitrogen, BMS618-3), and mouse TNF-α (Invitrogen, 88-7324-88) kits. The lactate dehydrogenase (LDH) activity in culture supernatants of the infected cells was measured using a CytoTox 96 assay kit (Promega), in accordance with the manufacturer’s protocol.
8
Cytotoxicity Quantification via LDH Assay
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LDH release was quantified using the Cytotox96 Assay Kit (Promega) according to the manufacturer's instructions. Cytotoxicity was normalized to Triton (100%) and LDH release from untreated cells was used for background subtraction.
9
Nasal Delivery of Exendin-4 in Rats
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The exendin-4/PTD mixtures prepared as described above were applied to the nostrils of anesthetized rats (dose of exendin-4, 30 µg/kg). Untreated rats served as negative controls. The positive control group was nasally administered to rats with 5% (w/v) sodium taurodeoxycholate. After 15 min, the nasal cavity was washed with 1 mL PBS using a micropipette. The washed solution was collected, and LDH activity in the wash solution was measured using a CytoTox-96 assay kit (Promega, Madison, WI) according to the manufacturer’s protocol. LDH leakage into the nasal fluid after the nasal administration of 5% (w/v) sodium taurodeoxycholate was defined as 100% leakage.
10
Cytotoxicity and Cytokine Profiling
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All the cultured supernatants mentioned above were collected at the indicated time in this study. Lactate dehydrogenase (LDH) was measured for the cytotoxicity assessment using the Cytotox96 assay kit (Promega, USA). Interlukin 1β (IL-1β) and IL-6 were tested using ELISA kits (BD Biosciences, USA).
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