Lactate dehydrogenase ldh cytotoxicity detection kit

Biocompatibility of the MQDs with HUVECs at 3 and 7 days was assessed using the Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit (MK401, Takara Bio). Briefly, HUVECs were plated on 96‐well plates and grown to 80% confluency. They were then treated with varying concentrations of MQDs and grown for 7 days in culture. At 3 and 7 days, media were taken from the wells for LDH assessment. Six replicates were included for each treatment condition. Additionally, cell proliferation at 7 days was assessed using the WST‐1 Cell Proliferation Assay kit (K304, BioVision Incorporated). Briefly, HUVECs were plated on 96‐well plates and grown to 80% confluency. The cells were then treated with varying concentrations of MQDs and grown for 7 days in culture and used for the WST‐1 assay according to manufacturer protocols. Five replicates were included for each treatment condition.

The experiment was conducted using Lactate dehydrogenase (LDH) Cytotoxicity Detection Kit (Takara Bio Inc, Kusatsu, Japan), according to a manufacturer’s protocol. Briefly, the Pterosin B-treated cells were incubated for 48 h then and 100 μL of cell-culture supernatant was transferred into optically clear 96-well flat bottom microtiter plate. To determine the LDH activity, 100 μL of reaction mixture was added and incubated for 30 min at room temperature. The absorbance at 490 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance value of assay medium as background control was subtracted from other values of test samples.

Cyclophosphamide was purchased from Baxter Oncology GmbH (Batch No. 5J078A, Halle, Germany). Guinea pig serum was purchased from Beijing Bersee Bio Co., Ltd. Mouse IL-18 ELISA kit was purchased from R&D Systems (Minneapolis, USA). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit was purchased from TaKaRa (Otsu, Japan). LC/MS grade formic acid and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). HPLC grade water was prepared from distilled water using a Milli-Q system (Millipore Laboratory, Bedford, MA). Rutin, quercetin, tricetin, myricitrin, kaempferol, myricetin, apigenin, and luteolin reference standards were obtained from Sigma-Aldrich (St Louis, MO).

Mouse hippocampal HT22 cells were maintained in 10-cm dishes containing DMEM supplemented with 10% fetal calf serum (Satoh et al., 2000 (link), 2003; Sasaki et al., 2011 (link), 2013 ). The cells were seeded onto 24-well plates at a density of 8 × 10⁴ cells per well in 500 μl of serum-containing DMEM. One hour after seeding, the cultures were incubated for 1 hr with vehicle (DMSO) or various concentrations of D1 or D3. One hour later, the cells were exposed to 5 mM glutamate for 24 hr to induce oxidative damage. Subsequently, viability of the HT22 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, as described elsewhere (Satoh et al., 2000 (link), 2003; Sasaki et al., 2011 (link), 2013 ). Cell death was also measured by the lactate dehydrogenase (LDH) cytotoxicity detection kit (TAKARA, Otsu, Shiga, Japan), which quantitatively measures the release of LDH into the medium following cell lysis or cell death. This assay was used as described by the manufacturer.

Chlorpropamide (CLP, >97% purity), tolbutamide (TLB, >97% purity), ascorbic acid (ASC, >99.0% purity), citric acid (CIT, ≥99.5% purity), p-aminobenzoic acid (PABA, 99.0% purity), tromethamine (TRIS, >99.0% purity), Triton^® X-100, thiazolyl blue tetrazolium (MTT), dimethyl sulfoxide (DMSO), and Dubelcco’s phosphate buffered saline, trypan-blue solution were acquired from Sigma-Aldrich (St. Louis, MO, USA). L-(+)-arginine (ARG, ≥98% purity) was purchased from Acros Organics (Fair Lawn, NJ, USA), L-tryptophan (TRY, 99% purity) from Alfa Aesar (Kandel, Germany), and malic acid (MAL, >99.5% purity) from Merck (Darmstadt, Germany). The water used was ultra-pure grade (resistivity of 18.2 MΩ·cm), purified by a Heal Force ultra-pure water system (Shanghai, China). All chemicals were used without further purification.
Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, trypsin-EDTA (1×), penicillin–streptomycin (PenStrep), inactivated fetal bovine serum (FBS), and non-essential amino acids were purchased from Gibco by Life Technologies (New York, NY, USA. A lactate dehydrogenase (LDH) cytotoxicity detection kit was obtained from Takara Bio Inc. (Shiga, Japan). Murine fibroblast (L929) and intestinal epithelial (Caco-2) cell lines were acquired from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK).

Supernatants from stimulation experiments were used fresh. Cytokine concentrations were measured by ELISAs according to the manufacturer’s instructions in Nunc MaxiSorp flat-bottom plates (Invitrogen) using Uncoated ELISA Kits (Invitrogen) for TNF-α, IFN-γ, and IL-2. Lactate dehydrogenase (LDH) Cytotoxicity Detection Kit (Takara Bio) was used as per manufacturer instructions to detect cell killing.

Cell viability was measured by the CCK-8 assay (Dogen, Seoul, Korea). Briefly, H9c2 cells were seeded at 1 × 10⁴ cells per well in a 96-well plate and treated with H₂O₂. After 3 h, 10 µL of CCK-8 reagent per well was added, and the cells were incubated for 1 h at 37 °C. For the cytotoxicity assay, a Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit (TaKaRa, Nojihigashi, Kusatsu, Shiga, Japan) was used. Cell culture supernatant was mixed with 100 μL of prepared LDH reagent in each well. The plate was incubated for 30 min at room temperature (RT). The optical density (OD) was measured at 450 nm with a microplate reader (Multiskan FC, Thermo Fisher Scientific).