NK cells were isolated for ADCC assay. Briefly, PBMCs were isolated from healthy donors by using a Ficoll-Conray gradient, followed by NK-cell isolation using a CD56 NK-cell isolation kit and a MACS LS column (Miltenyi Biotech) according to the manufacturer’s instructions. ADCC activity of NK cells was measured using a colorimetric CytoTox 96 assay (Promega). This system quantifies the release of lactate dehydrogenase (LDH) from target cells. NK cells were mixed with 2 × 104 target cells with or without mogamulizumab (2 µg/mL, antihuman CCR4 humanized mAb, Kyowa Hakko Kirin Co.) or cetuximab (2 µg/mL, antihuman epidermal growth factor receptor chimeric mAb, Merck AG) at different effector-to-target (E:T) ratios in 96-well round-bottomed plates. After 6 h incubation at 37 °C, 50 µL supernatants were collected and incubated with coloring agent at room temperature for 30 min. LDH density was measured using a fluorescence plate reader (490 nm).
Cytotox 96 assay
Manufactured by Promega
Sourced in United States
The CytoTox 96 assay is a colorimetric assay designed to quantify the release of lactate dehydrogenase (LDH) from damaged cells. It provides a straightforward method for determining the cytotoxicity of test compounds or other stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by GE Healthcare (2 mentions)
Polyethyleneimine, by Merck Group (2 mentions)
Sy sh5y cells, by American Type Culture Collection (2 mentions)
Aβ42q15a, by Genecust (2 mentions)
Aβ16q15a, by Genecust (2 mentions)
Biotinylated aβ42, by Genecust (2 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Hacat, by Lonza (1 mentions)
Celltiter96 assay, by Promega (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Chromate, by Awareness Technology (1 mentions)
Tissue culture plate, by BD (1 mentions)
Nk macs medium, by Miltenyi Biotec (1 mentions)
Annexin 5 apc and propidium iodide, by BD (1 mentions)
Caspase glo 3 7 reagent, by Promega (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Triton x 100, by Merck Group (1 mentions)
48 protocols using cytotox 96 assay
1
ADCC Assay for NK Cell Activity
2
Cytotoxicity Assay for T-cell-Mediated Lysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with or without 5-Aza for an additional 48 h. Viable T-cells were determined by trypan blue exclusion and 50.000 T-cells were cocultured with target cell lines (HL60 and K562) in different ratios for example, 1 : 5, 1 : 10, and 1 : 20. Cells were cocultured for 4 h and specific cytotoxicity was measured using the CytoTox 96 assay (Promega, Madison, USA), according to the manufacturer's instructions. In brief pretreated T-cells were cocultured with target cells, for additional 4 hours. The assay measures the released LDH in culture supernatants in a 30-minute coupled enzymatic assay, which results in conversion of a tetrazolium salt into a red formazan product. The amount of color formed is proportional to the number of lysed cells. As additional controls to untreated cells we used K562 cells for spontaneous lysis. Cell death was calculated according to the company provided formula. All cytotoxicity experiments were performed in triplicates and the mean value was used for further analysis.
3
Cytotoxicity Evaluation via LDH Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytotoxicity in different transfection conditions was assessed through LDH assay (#G1780, Cytotox 96®assay, Promega, Madison, WI, USA) according to the manufacturer’s instructions. The LDH absorbance signal was measured at 492 nm with an optical plate reader (FLUOstar galaxy). The maximum LDH release was assessed by incubating cells with 10 µL of Lysis buffer for 30 min prior to analysis. LDH absorbance was normalized to protein concentration, and data is presented as a percentage of NC siRNA.
4
Cytotoxicity Assessment of Bacteria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytotoxicity activity of bacteria was studied using the human keratinocyte cell line HaCaT (Eppelheim, Germany) as previously described (N’Diaye et al., 2016 (link)). Briefly, HaCaT cells were grown at 37°C under 5% CO2 atmosphere, in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Levallois-Perret, France) supplemented with 10% fetal calf serum and 1% antibiotic cocktail (HyClone Thermo Scientific, Illkirch, France). Cells were used between passages 41 and 65. One day before use, HaCaT cells were starved of antibiotic and fetal calf serum. Cells were incubated for 24 h with bacteria at a multiplicity of infection (MOI) of 10:1. Bacterial cytotoxicity was determined by measurement of lactate dehydrogenase (LDH) release by HaCaT cells. LDH is a stable cytosolic enzyme that diffuses into the culture medium upon cell lysis and was measured using a Cytotox 96 assay (Promega). Results are the mean of three independent experiments each done in independent triplicate measurement.
5
Quantifying Cell Cytotoxicity via LDH Release
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytosolic lactate dehydrogenase (LDH) release (OD490nm) was measured using CytoTox 96 assay (Promega) to monitor cytotoxicity. Lysing of infected cells with 1% Triton-X enabled maximum LDH release, while supernatants of control cells lysates were assessed for spontaneous LDH release. Measurement of cytotoxicity was carried out by calculating the percentage of LDH release net change as shown in the following formula: (test LDH release - spontaneous release)/(maximal release - spontaneous release) ×100.
6
Cell Viability and Membrane Permeability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was determined by MTS reagent using the CellTiter96 assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol with the below exception. In order to avoid artifacts in the optical density (OD) values, steps were taken to remove the MTS reagent (transferring it into another plate) to remove obstruction of cell/particle material during absorbance measurements at 490 nm. All Ag particle exposed cells were compared to the no particle control condition and expressed as percent viable cells relative to control condition. It is possible to have greater than 100% viability, which indicates cell proliferation. Cell membrane permeabilization in the macrophage models only was determined by release of lactate dehydrogenase (LDH) using the CytoTox96 assay (Promega, Madison, WI, USA) according to manufacturer’s protocol. Percent LDH release was expressed relative to 100% total lysed cells (cell lysis done 1 h before assay with provided reagent) at the same cell concentration.
7
Cytotoxicity Assay for OVA-Specific CTLs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OVA specific cytolytic activity in vitro was determined via lactate dehydrogenase (LDH) release CytoTox96 Assay (Promega, Madison, WI, USA) according to manufacturer’s recommendations. Briefly, effector lymphocytes were cultured in limiting dilution either alone or with appropriate target cells, EL4 or E.G7-OVA at 37 °C for 18 h. CTL activity was assessed by measuring relative LDH with maximum and spontaneous release values measured against LDH within supernatants of effector target combinations. Specific lysis was calculated as follows: percent specific lysis (%) = 100 × [(experimental – T cell sponta neous)/(target max – target spontaneous)].
8
Cell Viability Evaluation via LDH Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was evaluated by measuring lactate dehydrogenase (LDH) leakage from damaged cells due to the loss of cell membrane integrity. The activity of LDH release in culture medium was measured using the CytoTox 96® assay (Promega) according to manufacturer’s instructions. Briefly, the culture supernatant was incubated with substrate mix for 30 min in the dark at RT, followed by addition of stop solution. Absorbance was then recorded at 490 nm using an EnSpire® Multimode Plate Reader (Perkin-Elmer). Results are expressed as a percentage of maximum LDH release obtained by complete cell lysis.
9
LDH Cytotoxicity Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture media was assessed for lactate dehydrogenase (LDH) 24 h after treatment using the Cytotox 96 assay (Promega) according to the manufacturer’s instructions. Absorbance was measured using a ChroMate microplate reader (Awareness Technology Inc., Palm City, FL).
10
Cytotoxicity Assay for Methotrexate
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Cytotox96 assay from Promega (cat. no.G1781) is a colorimetric-based cytotoxicity assay that quantitatively measures the release of lactate dehydrogenase (LDH) from damaged cells.CytoTox 96 Non-Radioactive Cytotoxicity Assay was used following treatment with MTX 1μ M and 10μ M. After treatments, culture medium was recovered, and then cells were lyzed following the manufacturer's instructions. Released LDH in culture medium was measured for detection of cell damage following treatments. Intracellular LDH (induced by the addition of the lysis buffer) was measured for determination of the maximum LDH release. The percentage of cellular injury was calculated using the formula: % cytotoxicity = 100 × experimental LDH release / maximum LDH release.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!