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A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time.

ABSTRACT: Analyzing the effects on cell growth inhibition and/or cell death has been an important component of biological research. The MTS assay and LDH-based cytotoxicity assays are two of the most commonly used methods for this purpose. However, data here showed that MTS cell proliferation assay could not distinguish the effects of cell death or cell growth inhibition. In addition, the original LDH-based cytotoxicity protocol grossly underestimated the proportion of dead cells in conditions with growth inhibition. To overcome the limitation, we present here a simple modified LDH-based cytotoxicity protocol by adding additional condition-specific controls. This modified protocol thus can provide more accurate measurement of killing effects in addition to the measurement of overall effects, especially in conditions with growth inhibition. In summary, we present here a simple, modified cytotoxicity assay, which can determine the overall effects, percentage of cell killing and growth inhibition in one 96-well based assay. This is a viable option for primary screening for many laboratories, and could be adapted for high throughput screening.

SUBMITTER: Smith SM

PROVIDER: S-EPMC3219643 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time.

Smith Shilo M SM Wunder Michael B MB Norris David A DA Shellman Yiqun G YG

PloS one 20111117 11

Analyzing the effects on cell growth inhibition and/or cell death has been an important component of biological research. The MTS assay and LDH-based cytotoxicity assays are two of the most commonly used methods for this purpose. However, data here showed that MTS cell proliferation assay could not distinguish the effects of cell death or cell growth inhibition. In addition, the original LDH-based cytotoxicity protocol grossly underestimated the proportion of dead cells in conditions with growth ...[more]

PMID: 22125603

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Project description:Nanotechnologies such as nanoparticles are established components of new medical devices and pharmaceuticals. The use and distribution of these materials increases the requirement for standardized evaluation of possible adverse effects, starting with a general cytotoxicity screening. The Horizon 2020 project "Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE)" identified in vitro cytotoxicity quantification as a central task and first step for risk assessment and development for medical nanocarriers. We have performed an interlaboratory comparison on a cell-assay matrix including a kinetic lactate dehydrogenase (LDH) release cell death and WST-8 cell viability assay adapted for testing organic nanocarriers in four well-characterized cell lines of different organ origins. Identical experiments were performed by three laboratories, namely the Biomedical Technology Center (BMTZ) of the University of Münster, SINTEF Materials and Chemistry (SINTEF), and the National Institute for Public Health and the Environment (RIVM) of the Netherlands according to new standard operating procedures (SOPs). The experiments confirmed that LipImage™ 815 lipidots® are non-cytotoxic up to a concentration of 128 µg/mL and poly(alkyl cyanoacrylate) (PACA) nanoparticles for drug delivery of cytostatic agents caused dose-dependent cytotoxic effects on the cell lines starting from 8 µg/mL. PACA nanoparticles loaded with the active pharmaceutical ingredient (API) cabazitaxel showed a less pronounced dose-dependent effect with the lowest concentration of 2 µg/mL causing cytotoxic effects. The mean within laboratory standard deviation was 4.9% for the WST-8 cell viability assay and 4.0% for the LDH release cell death assay, while the between laboratory standard deviation was 7.3% and 7.8% for the two assays, respectively. Here, we demonstrated the suitability and reproducibility of a cytotoxicity matrix consisting of two endpoints performed with four cell lines across three partner laboratories. The experimental procedures described here can facilitate a robust cytotoxicity screening for the development of organic nanomaterials used in medicine.

Project description:BackgroundIn the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits.Methodology/findingsThis work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material.Conclusions/significanceThe cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices.

Project description:γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical evaluation.

Dataset Information

A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time.

Publications

A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time.

Similar Datasets

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