Validation and stability analysis of a modified lactate dehydrogenase (LDH) test method to be employed for an in vitro viable skin model

Bauhammer, I.; Sacha, Manuel; Haltner, Eleonore

doi:10.1016/j.heliyon.2019.e01618

Cited by 6 publications

(9 citation statements)

References 24 publications

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“…Various LDH isoenzymes are expressed in tissue-specific manners, and they have diagnostic values for detecting tissue damage [ 58 ]. For example, LDH-3, composed of 2 subunits of LDHA and LDHB, is mainly located in lung tissue, and it is directly correlated to pleural fluid.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, it has been proposed as a biomarker of lung tissue damage and pulmonary endothelial cell injury [ 59 ]. Especially in muscle damage, which induced by intensive physical performance, LDH-5, composed of 4 LDHA isoforms, is also detected in serum [ 58 ]. The use of LDH as a biomarker has been shown to be successfully applied in pharmacokinetic studies [ 60 ].…”

Section: Discussionmentioning

confidence: 99%

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The Oral Administration of Sanguisorba officinalis Extract Improves Physical Performance through LDHA Modulation

et al. 2021

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Muscle fatigue is induced by an acute or chronic physical performance inability after excessive physical activity often associated with lactate accumulation, the end-product of glycolysis. In this study, the water-extracted roots of Sanguisorba officinalis L., a herbal medicine traditionally used for inflammation and diarrhea, reduced the activities of lactate dehydrogenase A (LDHA) in in vitro enzyme assay myoblast C2C12 cells and murine muscle tissue. Physical performance measured by a treadmill test was improved in the S. officinalis-administrated group. The analysis of mouse serum and tissues showed significant changes in lactate levels. Among the proteins related to energy metabolism-related physical performance, phosphorylated-AMP-activated protein kinase alpha (AMPKα) and peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC-1α) levels were enhanced, whereas the amount of LDHA was suppressed. Therefore, S. officinalis might be a candidate for improving physical performance via inhibiting LDHA and glycolysis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Oral Administration of Sanguisorba officinalis Extract Improves Physical Performance through LDHA Modulation

et al. 2021

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“…105 µL polyethylene glycol (PEG) 400 were added to the daily collected samples from medium supernatant containing LDH before storage at −20 °C for up to 4 weeks until measurement. These conditions were chosen according to the results of a previous stability study (Bauhammer, Sacha & Haltner, 2019b).…”

Section: Methodsmentioning

confidence: 99%

“…2). The generally cell-based LDH release assay was modified and validated for its usage on tissue beforehand (Bauhammer, Sacha & Haltner, 2019b).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, it can be used for repeated measurements with the same skin specimen which is crucial for this study (Roche, 2016). As this assay is also in principle designed for cell culture, a modified version, adapted to tissue (Bauhammer, Sacha & Haltner, 2019a) and validated according to FDA (Food and Drug Administration, USA) guidelines (Bauhammer, Sacha & Haltner, 2019b), was used in the current study.…”

Section: Introductionmentioning

confidence: 99%

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Establishment of an in vitro model of cultured viable human, porcine and canine skin and comparison of different media supplements

Bauhammer

Sacha

Haltner

2019

PeerJ

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Transdermal drug delivery provides several advantages over conventional drug administration, such as the avoidance of first-pass metabolism and better patient compliance. In vitro research can abbreviate and facilitate the pharmaceutical development considerably compared to in vivo research as drug screening and clinical studies can be reduced. These advantages led to the development of corresponding skin models. Viable skin models are more useful than non-viable ones, due to the influence of skin metabolism on the results. While most in vitro studies concentrate on evaluating human-based models, the current study is designed for the investigation of both human and animal diseases. So far, there is little information available in the literature about viable animal skin cultures which are in fact intended for application in the veterinary and not the human field. Hence, the current study aims to fill the gap. For the in vitro viable skin model, specimens of human, porcine and canine skin were cultured over two weeks under serum-free conditions. To evaluate the influence of medium supplementation on skin viability, two different supplement mixtures were compared with basic medium. The skin specimens were maintained at a viability-level >50% until the end of the study. From the tested supplements, the addition of bovine pituitary extract and epidermal growth factor increased skin viability whereas hydrocortisone and insulin induced a decrease. This in vitro viable skin model may be a useful tool for the investigation of skin diseases, especially for the veterinary field.

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Fully automated viability and toxicity screening—A reliable all‐in‐one attempt

Liedtke,

Weiss,

Skifov

et al. 2024

Cancer Medicine

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BackgroundThe CRISPR/Cas9 technology is nowadays a common tool for genome editing to achieve new insights into, for example, diagnostics and therapeutics in cancer and genetic disorders. Cell proliferation and anticancer drug response studies are widely used to evaluate the impact of editing. However, these assays are often time‐consuming, expensive, and reproducibility is an issue. To overcome this, we developed a fast and cheap assay that combines a fully automated multispectral fluorescence microscopy platform with a nuclei staining and open‐source software analysis.MethodsHere, we generated different LEDGF/p75 model cell lines to validate the effect on proliferation and chemosensitivity. Therefore, a fast protocol for an optimized all‐in‐one attempt for cytotoxicity screenings and proliferation analysis of adherent cells in a 96‐well plate format was established using differential staining with two fluorescent dyes (Hoechst 33342 and propidium iodide) for live/dead cell discrimination. Subsequently, an automated cell nuclei count and analysis were performed using bioimage informatics.ResultsWith the new established assay technology, up to 50,000 cells/well can be detected and analyzed in a 96‐well plate, resulting in a fast and accurate verification of viability and proliferation with consistency of 98% compared to manual counting. Our screening revealed that LEDGF depletion using CRISPR/Cas9 showed a diminished proliferation and chemosensitivity independent of cell line origin. Moreover, LEDGF depletion caused a significant increase in 𝛾H2AX foci, indicating a substantial increase in DNA double strand breaks. LEDGF/p75 overexpression enhanced proliferation and chemoresistance underlining the role of LEDGF in DNA damage response.ConclusionIndependent of cancer cell type, LEDGF/p75 is a central player in DNA damage repair and is implicated in chemoresistance. Moreover, our automated fluorescence biosensor technology allowed fast and reliable data acquisition without any fixation or additional washing steps. Additionally, data analysis was implemented using the modular open‐source software that can be adapted as needed.

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Validation and stability analysis of a modified lactate dehydrogenase (LDH) test method to be employed for an in vitro viable skin model

Cited by 6 publications

References 24 publications

The Oral Administration of Sanguisorba officinalis Extract Improves Physical Performance through LDHA Modulation

The Oral Administration of Sanguisorba officinalis Extract Improves Physical Performance through LDHA Modulation

Establishment of an in vitro model of cultured viable human, porcine and canine skin and comparison of different media supplements

Fully automated viability and toxicity screening—A reliable all‐in‐one attempt

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