To asses FA cytotoxicity, HUVEC were cultured in 96-well plates to 90–100% confluence, then exposed to EtOH, BSA, or MBCD solubilized OA, 12-OH OA, 2-OH OA (Na+), me-OA or vehicle controls, in quadruplicate, for 4, 24, or 48 h. Cell supernatants were collected immediately following exposure for use with the Promega Cytotox-96 cytotoxicity assay (Promega Corporation, Madison, WI) to measure supernatant lactate dehydrogenase (LDH) activity release. In some experiments, cell viability was visualized with standard light microscopy techniques by exclusion of trypan blue dye.
Cytotox 96 cytotoxicity assay
Manufactured by Promega
Sourced in United States
The CytoTox 96 cytotoxicity assay is a colorimetric method for quantifying the release of lactate dehydrogenase (LDH) from damaged cells. It provides a simple and reliable way to measure cytotoxicity in a variety of cell types.
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Lab products found in correlation
Mouse il 1β duoset elisa, by R&D Systems (3 mentions)
Z lr amc fluorogenic peptide substrate, by R&D Systems (3 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (3 mentions)
Cathepsin b inhibitor 2 calbiochem, by Merck Group (2 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous proliferation assay, by Promega (1 mentions)
Novapath microplate reader, by Bio-Rad (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
Alamarblue assay, by Thermo Fisher Scientific (1 mentions)
Dynabeads untouched mouse cd8 t cell kit, by Thermo Fisher Scientific (1 mentions)
Anti caspase 1, by Abcam (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Ac yvad cmk, by Cayman Chemical (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Abcam (1 mentions)
23 protocols using cytotox 96 cytotoxicity assay
1
Evaluating Fatty Acid Cytotoxicity on HUVECs
2
Pulmonary Inflammation and Damage Evaluation
3
LDH Release Assay for Pyroptosis
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Lactate dehydrogenase (LDH) release has been perceived as a hallmark of pyroptosis. CytoTox 96 Cytotoxicity Assay (Promega, Madison, WI, USA) was used to detect LDH activity in the supernatants after the indicated treatments. The activity of released LDH is expressed as a proportion of the total LDH in the cell lysate.
4
FLS Proliferation Assay with RTD-1
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FLS were seeded at 3 × 103 cells/well in a 96-well TC treated plate (Greiner). Cells were allowed to adhere for 18 h and medium was replaced prior to addition of peptide or vehicle. RTD-1 was added to wells at final concentrations of 0–30 μg/ml in culture medium and incubated for 0, 24, or 48 h at 37°C in 5% CO2. FLS cell number was determined by DNA staining using CyQuant Cell Proliferation Assay Kit (Invitrogen) per manufacturer’s instructions. Cellular toxicity was determined by measuring the release of lactate dehydrogenase (LDH) using a CytoTox 96 Cytotoxicity Assay (Promega Bio Sciences, San Luis Obispo, CA) in FLS supernatants. All assays were performed with ≥ 7 replicates.
5
Cell viability and cytotoxicity assay
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Cells were seeded in a 96-multiwell at a density of 10 000 cells per well. After 24 h, correctors dissolved in 1‰ DMSO were added at the indicated concentrations. After 24 h of treatment, cell viability was determined measuring the bioreduction of MTS (Owen’s reagent) by living cells with the Cell Titer 96 Aqueous proliferation assay (Promega) according to the manufacturer’s instruction. Cell toxicity was determined measuring the release of LDH in culture supernatant with the Cytotox 96 cytotoxicity assay (Promega) according to the manufacturer’s instruction. A maximum LDH release control was performed adding a lysis solution 45 min before adding Cytotox 96 reagent, and LDH release was calculated as (experimental LDH release-blank)/(maximum LDH release-blank)×100.
All spectrophotometric measurements were performed with a Novapath Microplate Reader (Bio-Rad). All experiments were run in sextuplicate.
All spectrophotometric measurements were performed with a Novapath Microplate Reader (Bio-Rad). All experiments were run in sextuplicate.
6
Cytotoxicity Quantification via LDH Assay
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Culture supernatants were collected and centrifuged at 250×g for 5 min after treatments. The LDH concentration was measured with the CytoTox 96 cytotoxicity assay (G1780, Promega, USA) at 490 nm according to the protocol offered by the manufacturer. The percentage of LDH release was calculated as follows: (Sample − Background)/(Maximum − Background) × 100%. Each subject was repeated at least three times.
7
Cell Viability Assay with TGFβ-1 and E64d
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To determine cell viability 1.5×104 cells were seeded in 9.6 cm2 wells. After two days cells were washed three times with DPBS and switched to indicator free growth medium containing 5% FCS supplemented with or without TGFβ-1+/−E64d in triplicates. Cell viability was assayed by LDH activity using the Cytotox 96® cytotoxicity assay (Promega) according to the manufacturer’s instructions. To compile growth curves, every two days cells of three wells of one condition were pooled, diluted in Trypan blue stain and living cells were counted using a Neubauer counting chamber.
8
Quantifying Lung Damage and Vascular Leakage
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To assess lung damage, bronchoalveolar lavage fluid was analyzed by measuring lactate dehydrogenase levels using a CytoTox 96 Cytotoxicity Assay (Promega) following the manufacturer’s instructions. To assess vascular/pulmonary leakage, bronchoalveolar lavage fluid was analyzed using an Albumin BCG Reagent Set (Eagle Diagnostics). A standard curve was made by diluting the calibrator in PBS. Then 100 μl of sample or standard was transferred to a 96 well flat-bottomed plate, mixed with 100 μl of BCG reagent, let sit at RT for 5 min and then read on a plate reader at 630 nm.
9
Quantifying Lung Damage and Vascular Leakage
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To assess lung damage, BALF from 72 h post-inoculation was analyzed by measuring lactate dehydrogenase levels using a CytoTox 96® Cytotoxicity Assay (Promega) following the manufacturer’s instructions. To assess vascular/pulmonary leakage, BALF was analyzed using an Albumin BCG Reagent Set (Eagle Diagnostics, Cedar Hill, TX, United States). A standard curve was made by diluting the calibrator in PBS/EDTA. Then 100 μl of sample or standard was transferred to a 96 well flat-bottomed plate, mixed with 100 μl of BCG reagent, let sit at RT (room temperature) for 5 min and then read on a plate reader at 630 nm.
10
Quantifying Parasite Cytotoxicity
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Parasites suspended in Ringer's solution at 5 × 105 parasites/ml were pretreated with varying concentrations of zaprinast (as indicated) for 20 min at 37 °C with 5% CO2. 105 parasites/well were then applied to confluent host cell monolayers, and plates were centrifuged at 290 × g for 5 min. Following 1 h at 37 °C with 5% CO2, plates were centrifuged again at 500 × g for 5 min. 50 μl of supernatant were collected from each well, and lactate dehydrogenase was quantified using the CytoTox 96 cytotoxicity assay (Promega).
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