Ldh cytotoxicity detection kit

Damage to the human colon epithelial cell line HT-29 (ATCC; HTB-38) was assessed using a lactate dehydrogenase (LDH) cytotoxicity detection kit (Sigma), which measures the release of LDH in the growth medium. The manufacturer’s protocol was followed. HT-29 cells were grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS to 95% confluence in a 96-well tissue culture plate and incubated at 37°C with 5% CO₂. Prepared cells were infected with 2 × 10⁶C. albicans SC5314 blastospores for 24 h at 37°C with 5% CO₂. Following incubation, 100 µl supernatant was removed from each experimental well and LDH activity in this supernatant was determined by measuring the absorbance at 490 nm (OD₄₉₀). LDH activity was calculated as the mean from at least four independent biological replicates.

Primary human chondrocytes of three different patients were seeded at 30% confluency with 3×10³ cells per well in 48-well plates. After 24 h in the expansion medium, cells were treated with minimal medium containing LG-DMEM, 2% human male AB serum (Merck KGaA, Darmstadt, Germany), 1% PenStrep, 1% GlutaMAX, and different concentrations of TNF-α (recombinant human TNF-α, reference 55418, BD Biosciences, Franklin Lakes, NJ, USA) or IFN-γ (animal-free recombinant human IFN-γ, reference AF-300-02, PeproTech, Thermo Fisher Scientific Inc) (0, 1, 10, and 100 ng/ml, respectively). Proliferation, metabolic activity, and lactate dehydrogenase (LDH cytotoxicity detection kit, reference 4744934001, Sigma-Aldrich, St. Louis, MO, USA) release were determined after 7 days upon stimulation as described below.

The membrane integrity of NIH3T3 fibroblasts was evaluated using an LDH Cytotoxicity Detection Kit (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were exposed to various concentrations of AgNPs for 24 h.

The membrane integrity of SH-SY5Y cells was evaluated using a LDH cytotoxicity detection kit (Sigma-Aldrich, St.Loius, MO, USA). A dead-cell protease activity assay was performed according to the method described earlier [34 (link)].

The cytotoxicity of PH to THP-1 or HT-29 cells was assessed by measuring lactate dehydrogenase (LDH) activity using an LDH cytotoxicity detection kit (Sigma-Aldrich, Saint Quentin Fallavier, France). The assay measures membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. Briefly, THP-1 or HT-29 cells were prepared as described previously. After 3 h treatment of cells with either LPS, LPS and peptides or LPS and dexamethasone, the supernatants were collected and cytotoxicity was evaluated by measuring LDH activity according to the manufacturer’s instructions. The absorbance was read at 490 and 650 nm, respectively. Control corresponded to supernatants from cells which had not undergone any treatment.

Cell viability was assessed using an LDH cytotoxicity detection kit (Sigma-Aldrich) per the manufacturerʼs protocol. In brief, WT or Trem2^−/− BV2 cells were plated in 96-well plates and cultured overnight either in media alone or in media with 20 µg ml⁻¹ or 40 µg ml⁻¹ cholesterol. The next day, supernatants were transferred to a new 96-well plate, along with wells of lysed cells and media alone for positive and negative controls. Samples were then treated with 100 µl of the reaction mixture and incubated at room temperature for 20 min. Then, samples were treated with 50 µl of stop solution, and absorbance was immediately recorded on a SpectraMax Plus plate reader at 492 nm. Percent cytotoxicity was determined using the absorbance values minus the background controls and normalized to baseline per the manufacturer’s instructions (% cytotoxicity = (sample value − negative control) / (positive control − negative control) × 100). For ER stress and cytotoxicity studies with phenylbutyrate (Sigma-Aldrich), cells were cultured overnight in 10 μM PBA or vehicle before analysis. For LXR agonist studies, cells were cultured overnight with the LXR agonist T0901317 (Sigma-Aldrich) at 10 μM.