Hla-/LukED-mediated cytotoxicity assay is performed as previously described.59 (link) Corresponding purified human or mouse antitoxin antibodies were diluted in PBS in 96-well round-bottom plates and incubated with 0.25 μg/mL recombinant Hla (Sigma) or 0.375 μg/mL LukE/LukD (Mayflower Bioscience) for 15 min at 37°C, then 2x105 THP-1 monocytes were added. Following static incubation for 4 h at 37°C with 5% CO2, the remaining non-lysed cells were pelleted, and the supernatants were transferred into a new 96-well flat-bottom plate. Cytolysis-mediated LDH release was quantified using commercially purchased LDH detection kit (Takara).
Ldh detection kit
Manufactured by Takara Bio
Sourced in Japan
The LDH Detection Kit is a laboratory assay used to quantitatively measure the activity of the enzyme lactate dehydrogenase (LDH) in biological samples. LDH is an important enzyme involved in cellular metabolism and its levels can provide insights into various physiological and pathological conditions.
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10 protocols using ldh detection kit
1
Hla-/LukED-mediated cytotoxicity assay
2
LDH Leakage Assay in Stressed AC16 Cells
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Transfected AC16 cells, treated with Sev for 1 h and H/R for 12 h, were seeded in 96-well plates (2 × 104 cells/well). Then, after collecting the cell supernatant, 60 μl of LDH detection solution from an LDH Detection Kit (catalog number: MK401; Takara Bio, Tokyo, Japan) was added to 150 μl of cell supernatant for further incubation at 37°C in the dark. About 30 min later, mass LDH leakage in the cell supernatant was measured at 490 nm with the LDH Detection Kit following the manuals of the producer.
3
Cytotoxicity Evaluation by LDH Assay
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Cytotoxicity was measured by evaluating the levels of lactate dehydrogenase in the cell culture supernatant using the lactate dehydrogenase (LDH) detection kit (Takara Bio, Inc., Tokyo, Japan) following the manufacturer’s protocol. LDH activity was detected by measuring absorbance at 490 nm using a microplate reader (Spectra Max M2, Molecular Devices, Sunnyvale, CA, USA).
4
Quantifying Oxidative Stress-Induced Cell Death
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LDH release assay, a well-known indicator of cell membrane integrity and viability, was used to examine the cell death induced by oxidative stress. The detailed protocol was followed based on the manufacturer’s instructions provided in the LDH detection kit (Takara). Between 36 and 48 h after infection with lentiviral particles, oxidative stress was induced by exposing the cells to 2 mM H2O2 for 3 h, as optimized based on previous studies [28 (link),29 (link)]. An amount of 100 μL of the supernatant was collected, centrifuged at 250× g for 5 min, and mixed with an equal volume of pre-prepared solution (catalyst/dye 1:45) for 30 min at room temperature in a 96-well plate. The absorbance of samples at 490 nm was measured using a Bio-Rad iMark microplate reader. The percentage of LDH release for each sample was normalized according to the absorbance reading from low-control samples that were treated with plain F-12 serum-free media and high-control samples that were treated with 0.5% Triton X-100.
5
Cytotoxicity Assessment of Quercetin Nanoparticles
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Lactate dehydrogenase (LDH) assays were conducted in order to evaluate cytotoxicity and nanoparticles potential for damaging cell membranes. LDH is an enzyme that is released from cells to the surrounding cell culture supernatant when cell membranes are damaged or disrupted. Cells were seeded in 96-well plates (10 4 cells per well) precoated with type I rat collagen. After 20 h of incubation at 37°C and 5% CO 2 , different concentrations of quercetin-loaded nanoparticles (functionalized and nonfunctionalized) were incubated with the cells for 4 h. EndoGRO medium and TX-100 sample were also used as negative and positive controls for cytotoxicity, respectively. After the incubation time, the medium of each well was collected and centrifuged (250 g for 10 min, RT) and the supernatant separated for further LDH quantification assay (LDH detection kit, Takara Bio Inc., Shiga, Japan).
After treatment with catalyst and dye solutions for 20 min at RT in the dark, absorbance was read at 490 and 690 nm using a Synergy™ HT Multi-mode Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA). Cytotoxicity was expressed as a percentage and compared to the maximum cytotoxicity of TX-100 sample.
After treatment with catalyst and dye solutions for 20 min at RT in the dark, absorbance was read at 490 and 690 nm using a Synergy™ HT Multi-mode Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA). Cytotoxicity was expressed as a percentage and compared to the maximum cytotoxicity of TX-100 sample.
6
Cytotoxicity Assay using LDH
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105 cells/well were stimulated in round bottom 96 well-plates in a volume of 200 μL. 100-μL of supernatant from each well was transferred into flat-bottom 96-well plates, then catalyst and dye solutions were added (LDH Detection Kit, Takara). Then 100 μL HCl 1N/well were added, and plates were read on a microplate reader (Multiskan, Thermo Scientific) at 450 nm. Results are given as % of maximum release, with medium = no cells and TX-100 = cells + 1% Triton X-100, finally % of maximum release = (OD sample − OD medium)/(OD TX-100 − OD medium) ×100.
7
Rat MAPC Membrane Damage Assay
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The assay for cell membrane damages was modified from our established assay for measuring rhMG53 activity in HEK293 cells [32 ,35 (link)]. In brief, rat MAPCs (1 × 106 cells/ml) were treated with extracellular rhMG53 or control protein (BSA), and then exposed to mechanical plasma membrane damage by orbital shaking with micro-glass beads. The level of membrane damage was measured by LDH release from the cells using a LDH Detection Kit (Takara Mountain View, CA, USA) at 490 nm as per manufacturer's instruction.
8
Real-time Cell Growth Monitoring
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Growth curves were determined by plating the cells (20%–30% confluence) in an xCELLigence System (Roche, Basel, Switzerland), which allows for automated, noninvasive, and real-time monitoring of live cells in culture. Biochemical analysis for necrotic cell death was performed by measuring lactate dehydrogenase (LDH) activity in culture media (LDH Detection Kit; Takara Bio).
9
LDH Cytotoxicity Assay Protocol
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Cytotoxicity was measured by evaluating the levels of lactate dehydrogenase in the cell culture supernatant using the lactate dehydrogenase (LDH) detection kit (Takara Bio, Inc., Tokyo, Japan) following the manufacturer's protocol. LDH activity was detected by measuring absorbance at 490 nm using a microplate reader (Spectra Max M2, Molecular Devices, Sunnyvale, CA, USA).
10
LDH Cytotoxicity Assay Protocol
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Cytotoxicity was measured by evaluating the levels of lactate dehydrogenase in the cell culture supernatant using the lactate dehydrogenase (LDH) detection kit (Takara Bio, Inc., Tokyo, Japan) following the manufacturer's protocol. LDH activity was detected by measuring absorbance at 490 nm using a microplate reader (Spectra Max M2, Molecular Devices, Sunnyvale, CA, USA).
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