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Journal ArticleDOI

In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride.

05 Jan 2006-Toxicology Letters (Elsevier)-Vol. 160, Iss: 2, pp 171-177
TL;DR: Different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.
About: This article is published in Toxicology Letters.The article was published on 2006-01-05. It has received 1305 citations till now. The article focuses on the topics: MTT assay & Cytotoxicity.
Citations
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Journal ArticleDOI
TL;DR: The data demonstrates that ZnO NPs even at low concentrations possess a genotoxic potential in human epidermal cells which may be mediated through lipid peroxidation and oxidative stress, Hence, caution should be taken in their use in dermatological preparations as well as while handling.

573 citations

Journal ArticleDOI
TL;DR: Technological developments are now enabling multidisciplinary approaches including molecular dynamics simulations combined with biophysics and microbiology toward providing valuable insights into the interactions of AMPs with membranes at atomic level, and has begun to contribute meaningfully toward the discovery of new AMPs.
Abstract: Antimicrobial peptides (AMPs) are promising next generation antibiotics that hold great potential for combating bacterial resistance. AMPs can be both bacteriostatic and bactericidal, induce rapid killing and display a lower propensity to develop resistance than do conventional antibiotics. Despite significant progress in the past 30 years, no peptide antibiotic has reached the clinic yet. Poor understanding of the action mechanisms and lack of rational design principles have been the two major obstacles that have slowed progress. Technological developments are now enabling multidisciplinary approaches including molecular dynamics simulations combined with biophysics and microbiology toward providing valuable insights into the interactions of AMPs with membranes at atomic level. This has led to increasingly robust models of the mechanisms of action of AMPs and has begun to contribute meaningfully toward the discovery of new AMPs. This review discusses the detailed action mechanisms that have been put forward, with detailed atomistic insights into how the AMPs interact with bacterial membranes. The review further discusses how this knowledge is exploited towards developing design principles for novel AMPs. Finally, the current status, associated challenges and future directions for the development of AMP therapeutics are discussed.

366 citations


Cites background from "In vitro cytotoxicity assays: compa..."

  • ...Cellular toxicity refers to single cell toxicity, which can be measured for human cells using MTT assays, LDH release, and ATP synthesis (Fotakis and Timbrell, 2006)....

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  • ...Cellular toxicity refers to single cell toxicity, which can be measured for human cells using MTT assays, LDH release, and ATP synthesis (Fotakis and Timbrell, 2006)....

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Journal ArticleDOI
TL;DR: The MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected, and the SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.
Abstract: The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated. Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC50), while IC50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays. This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.

345 citations


Additional excerpts

  • ...Cell enumeration independent of enzymati conversion of dye [30,31]...

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Journal ArticleDOI
TL;DR: This review collected the available data from cytotoxicity, oxidative stress and hemocompatibility studies in vitro and analysed their outcomes and provided a summary of the availableData in a form of reference table.

328 citations


Cites result from "In vitro cytotoxicity assays: compa..."

  • ...These findings are perfectly in agreement with previous comparisons of various cell viability assays [24]....

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Journal ArticleDOI
TL;DR: There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents, and tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.
Abstract: Some cancer patients are immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, increasing evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. We have assessed the expression of human leucocyte antigen class 1 (HLA1) on tumour cells before and after chemotherapy agents (cyclophosphamide, oxaliplatin or gemcitabine). In addition, we show that chemotherapy-stressed tumour cells may release cytokines that enhance the interactions between dendritic cells (DCs) and T cells into growth media. Here we report that some chemotherapy agents can increase HLA1 expression in tumour cells, even when expression is low. Increases were associated with killing by cytotoxic T cells, which were negated by HLA1-blockade. Furthermore, T-cell function, as indicated by increased proliferation, was enhanced as supernatants derived from tumours treated with chemotherapy augmented DC-maturation and function. There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.

283 citations


Cites methods from "In vitro cytotoxicity assays: compa..."

  • ...The discrepancy between, and limitations in, the assays have been noted previously (Lobner, 2000; Fotakis and Timbrell, 2006; Liu and Dalgleish, 2009) and could be because of the washing steps that are part of the MTT assay, whereas the LDH assay uses the culture medium directly from the cells....

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References
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Journal ArticleDOI
TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.

225,085 citations

Journal ArticleDOI
TL;DR: A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation and is used to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.

50,114 citations


"In vitro cytotoxicity assays: compa..." refers methods in this paper

  • ...The MTT assay was tested for its validity in various cell lines (Mossmann, 1983)....

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  • ...The MTT assay is based on the protocol described for the first time byMossmann (1983) ....

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  • ...Further modification of the initial protocol described by Mossmann (1983)were proposed (Denizot and Lang, 1986; Hansen et al., 1989) in order to improve the repeatability and the sensitivity of the assay....

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Journal ArticleDOI
TL;DR: The reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays.

4,631 citations


"In vitro cytotoxicity assays: compa..." refers methods in this paper

  • ...Further modification of the initial protocol described by Mossmann (1983) were proposed ( Denizot and Lang, 1986; Hansen et al., 1989) in order to improve the repeatability and the sensitivity of the assay....

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  • ...Further modification of the initial protocol described by Mossmann (1983)were proposed (Denizot and Lang, 1986; Hansen et al., 1989) in order to improve the repeatability and the sensitivity of the assay....

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Journal ArticleDOI
TL;DR: It was shown that phenol red does not interfere with the measurements and no washing steps are required since all ingredients can be added subsequently, and Serum proteins at concentrations up to 25% have no influence on the result.

3,442 citations

Journal ArticleDOI
TL;DR: Most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors.

1,331 citations


"In vitro cytotoxicity assays: compa..." refers background in this paper

  • ...More recent evidence suggests that reduction of MTT can also be mediated by NADH or NADPH within the cells and out of mitochondria (Berridge and Tan, 1992)....

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  • ...Keywords: Cadmium; LDH; MTT; Neutral red; Cytotoxicity...

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