The HCC cell lines SNU398, SNU387, Huh7, HepG2, and PLC/PRF/5 were obtained from ATCC. Cells were grown in Roswell Park Memorial Institute (RPMI) medium or Dulbecco’s Modified Eagle’s Medium (DMEM); supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin; and maintained at 37°C with a 5% CO2 atmosphere. Regorafenib (#CS-1205) and sorafenib (#CS-0164) were purchased from ChemScene. Lenvatinib (#19375) and cabozantinib (#18464) were purchased from Cayman Chemical. Trametinib (#S-2673) was purchased from Selleck Chemicals, and buparlisib (#HY-70063) was from MedChem Express. Recombinant human IFNγ (#300-02) was obtained from PeproTech. Cell viability was quantified with Cell Count Reagent SF (nacalai tesque). Absorbance at 450 nm was measured on a micro- plate reader. For quantification of cytotoxicity, we used LDH Cytotoxicity Assay Kit (nacalai tesque). The absorbance value at 490 nm was measured.
Ldh cytotoxicity assay kit
Manufactured by Nacalai Tesque
Sourced in Japan
The LDH Cytotoxicity Assay Kit is a colorimetric assay that quantifies the amount of lactate dehydrogenase (LDH) released into the cell culture medium upon cell lysis or damage. It provides a simple and reliable method to measure cytotoxicity or cell viability in various experimental models.
Automatically generated - may contain errors
Lab products found in correlation
Imark microplate reader, by Bio-Rad (2 mentions)
Recombinant human ifn γ, by Thermo Fisher Scientific (1 mentions)
Plc prf 5, by American Type Culture Collection (1 mentions)
Snu 387, by American Type Culture Collection (1 mentions)
Snu398, by American Type Culture Collection (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Cabozantinib, by Cayman Chemical (1 mentions)
Trametinib, by Selleck Chemicals (1 mentions)
Regorafenib, by ChemScene (1 mentions)
Lenvatinib, by Cayman Chemical (1 mentions)
Buparlisib, by MedChemExpress (1 mentions)
Sorafenib, by ChemScene (1 mentions)
Mouse tnf α quantikine elisa kit, by R&D Systems (1 mentions)
Pei max, by Polysciences (1 mentions)
Silencer select, by Thermo Fisher Scientific (1 mentions)
Viromer blue transfection reagent, by BioNTech (1 mentions)
Rhifn γ, by R&D Systems (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Stereomicroscope, by Olympus (1 mentions)
18 protocols using ldh cytotoxicity assay kit
1
Cytotoxicity Evaluation of Anti-Cancer Drugs
2
Evaluating TNF-α Production and Cytotoxicity
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RAW264.7 cells were seeded into cell culture plates. For immunofluorescence imaging, coverslips were plated and the cells were seeded onto coverslips. The plasmid constructs were transfected into cells using PEI-Max (Polysciences, Inc., Warrington, PA, United States) according to the manufacturer’s instructions. To examine TNF-α production by the transfected cells, cell culture supernatants were collected 24 h after transfection. TNF-α levels in the culture supernatants were measured using a mouse TNF-α Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, United States) according to the manufacturer’s instructions. For treatment with recombinant proteins, 10 μg/ml or the indicated concentrations of recombinant proteins purified from E. coli were added to culture supernatants for the indicated durations. Control green fluorescent protein (GFP) were boiled to denature state before treatment for avoiding unexpected effect. Lactate dehydrogenase (LDH) release in culture supernatants were measured using a LDH Cytotoxicity Assay Kit (Nacalai) according to the manufacturer’s instructions.
3
Macrophage Kynureninase Silencing Assay
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Macrophages were stimulated with recombinant human interferon-γ (rhIFN-γ; R＆D Systems, Minneapolis, MN, USA) at a final concentration of 10 ng/mL in serum- and antibiotic-free RPMI1640 for 24 h. Macrophages were transfected by incubating human kynureninase siRNA (Silencer® Select, siRNA ID; s17103 and/or s17104; Life Technologies, Carlsbad, CA, USA) or negative control siRNA (Silencer® Select, Negative Control #2 siRNA; Life Technologies) at a final concentration of 37.5 nM under the serum- and antibiotic-free RPMI1640 for 24 h, using Viromer® Blue transfection reagent (Lipocalyx). To examine siRNA-induced cell damage, lactate dehydrogenase (LDH) in the medium was measured using the LDH cytotoxicity assay kit (Nacalai Tesque, Kyoto, Japan). First, 100 µL of the supernatant from each well in which macrophages had been cultured under the indicated conditions was transferred to an optically transparent 96-well plate. A total of 100 µL of substrate solution was added and incubated for 20 min at RT under protection from light to each well of the above plate. Finally, 50 µL of stop solution was added to each well, and the absorbance was measured at 490 nm using a microplate reader. The results are presented as absorbance values and expressed as mean±standard deviation (SD).
4
Evaluating Extracellular Vesicle Cytotoxicity and Cytokine Activity
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A total of 10 µg of EV solution was treated with dTHP-1 cells, which were seeded at 1 × 105 cells/well in 24-well plates and differentiated for 72 h. After incubation for 4 h in RPMI1640 supplemented with 5% FBS at 37°C in 5% CO2 atmosphere, the supernatants were used for cytotoxicity and cytokine activity assays.
The cytotoxicity assay was performed using a lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Nacalai Tesque) according to the manufacturer’s instructions, and the release of LDH was measured by the absorbance at 450 nm by using an iMark microplate reader (Bio-Rad). For the assessment of cytokine activity, interleukin (IL)-8, tumor necrosis factor (TNF)-α, and IL-1beta were quantified using a human IL-8/TNF-α/IL-1beta ELISA kit (Proteintech) according to the manufacturer’s instructions.
The cytotoxicity assay was performed using a lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Nacalai Tesque) according to the manufacturer’s instructions, and the release of LDH was measured by the absorbance at 450 nm by using an iMark microplate reader (Bio-Rad). For the assessment of cytokine activity, interleukin (IL)-8, tumor necrosis factor (TNF)-α, and IL-1beta were quantified using a human IL-8/TNF-α/IL-1beta ELISA kit (Proteintech) according to the manufacturer’s instructions.
5
Cytotoxicity Evaluation of Benz Treatment
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The cytotoxicity was measured using the index of lactate dehydrogenase (LDH) release from cells and expressed as a percentage of the total cellular activity. LuM1 cells were seeded at 5000 cells/well in 96-well NCP and cultured with or without Benz. The culture medium was transferred to the other 96-well plates at 24 h, 48 h, and 72 h after Benz addition. The LDH activity was measured using the LDH cytotoxicity assay kit according to the manufacturer’s instructions (Nacalai Tesque, Kyoto, Japan) by measuring the absorbance (490 nm).
6
Cytotoxicity Assay for Alpha-Synuclein
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Cytotoxic effects of HMW- and LMW-αS were also inferred from the amount of LDH released from cells with damaged membranes using an LDH Cytotoxicity Assay kit (Nacalai Tesque, Inc.) according to the manufacturer’s instructions. After exposure of cells to HMW-αSo or LMW-αS for 24 h, the formazan product produced by LDH and released into the medium was measured at a wavelength of 490 nm using a Spectra Max i3 instrument (Molecular Devices).
7
Cytotoxicity Assay of FAP Compounds
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HUEhT-1 cells were incubated for 24 h with a culture medium containing FAP or FAP (Proemend) at different concentrations at follows: FAP; 0, 1, 3, 10, 30, 50, 100 µg/ml; FAP (Proemend); 0, 1.5, 4.5, 7.5, 15, 30, 45, 150 µg/ml. After 24 h-incubation, the concentrations of lactate dehydrogenase (LDH) leaked from the cells into the incubation medium were determined using LDH Cytotoxicity Assay Kit (Nacalai Tesque, Kyoto, Japan). Separately, cells incubated with a culture medium alone were mixed with Triton-X (final concentration, 1%) to estimate the maximal leakage of LDH (100%).
8
Bexarotene Cytotoxicity Evaluation in Cultured Cells
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In the culture medium, we seeded the cells in 12-well plates (100,000 cells/well). At 80% confluence, the cells were treated with 0–20 µM of bexarotene (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) dissolved in dimethyl sulfoxide (Fujifilm Wako, Osaka, Japan) for 24 h. Bexarotene is insoluble in water; hence, it was dissolved in dimethyl sulfoxide (DMSO). Therefore, all the experiments, including the untreated control, were performed with a concentration of 0.1% DMSO. The cells were then treated with trypsin–EDTA solution (Nacalai Tesque) and mixed with the total amount of trypan blue solution (Nacalai Tesque). Subsequently, the live cells were counted using a cell-counting chamber (WakenBtech Co., Ltd., Kyoto, Japan) under a stereomicroscope (Olympus, Tokyo, Japan).
Moreover, we collected the cell culture supernatant and measured the LDH activity using the LDH Cytotoxicity Assay Kit (Nacalai Tesque) according to the manufacturer’s protocol. The LDH levels of the cell culture supernatant derived from the bexarotene-treated cell group were normalized relative to the cultured medium derived from the control group (vehicle of the bexarotene-treated group). Each experiment was repeated at least three times, and the LDH activity was compared to that of the control group.
Moreover, we collected the cell culture supernatant and measured the LDH activity using the LDH Cytotoxicity Assay Kit (Nacalai Tesque) according to the manufacturer’s protocol. The LDH levels of the cell culture supernatant derived from the bexarotene-treated cell group were normalized relative to the cultured medium derived from the control group (vehicle of the bexarotene-treated group). Each experiment was repeated at least three times, and the LDH activity was compared to that of the control group.
9
Cytotoxicity Assessment via LDH Release
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Cytotoxicity was measured using the index of lactate dehydrogenase (LDH) release from cells, as described previously [10 ]. The LDH activity was measured using the LDH cytotoxicity assay kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions. The culture supernatants of cells transfected with siRNA were collected at 48 h after the replacement of media (72 h after siRNA transfection). The collected supernatants were transferred to another 96-well plate and incubated with substrate solution at RT for 20 min. The cells were lysed with cell lysis buffer, then incubated with substrate solution at RT for 20 min. The stop solution was added and absorbance (490 nm) was measured. Each absorbance was converted into a relative value to the control. For normalization, the relative level of LDH release per cell number was calculated.
10
Quantifying Drug Cytotoxicity via LDH Assay
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The cytotoxicity of the drug treatment was quantified by measuring lactate dehydrogenase (LDH) activity released into the culture medium using an LDH cytotoxicity assay kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s protocol. In brief, 100 µL culture supernatants in a 96-well plate were added with 100 µL substrate solution. The plate was protected from light and incubated for 20 min at room temperature. Following the addition of 50 µL stop solution, absorbance was measured at 490 nm using an iMark microplate reader. Measured values were normalized to the value of vehicle control.
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