Current in vivo Models for Brain Disorders
Carla Vitorino, Andreia Jorge, Alberto Pais in Nanoparticles for Brain Drug Delivery, 2021
In the specific case of brain tumours, one of the most utilised techniques to visualise drug delivery into the tumour is using BLI. The firefly luciferase (FLuc) can be used as a reporter gene and d-luciferin as a substrate. FLuc catalyses d-luciferin oxidation in the presence of adenosine triphosphate (ATP) and coenzyme A (CoA) producing O2 and photons which can be detected in vivo. Recording of the emitted light by photodetectors, capable of linear response allows for real-time measurements [57, 58]. This technique is based on the stable expression of luciferase by the brain tumour cell line selected for the experiment. Once the cells are implanted into the mouse brain, d-luciferin could be injected intraperitoneally into the host mice, where it is distributed throughout the mouse body and crosses the BBB. The intensity of the emitted light correlates to the size of the tumour and allows comparisons of tumours size across different animals but also within the same animal in different time points (Fig. 13.1) [59].
Lab-on-a-Chip-Based Devices for Rapid and Accurate Measurement of Nanomaterial Toxicity
Suresh C. Pillai, Yvonne Lang in Toxicity of Nanomaterials, 2019
Bioluminescence and chemiluminescence represent similar phenomena. Because of a series of chemical reactions, reactive compounds give rise to a molecule which is in an electronically excited state. Ultimately, the excited molecule returns to its ground states (lower energy level) and emits characteristic photons of light. In fact, bioluminescence is a subcategory of chemiluminescence, where the source of emission is a bio-molecule. For example, a class of different bio-compounds called Luciferin are the source of bioluminescence in several living organisms. Light-emitting Luciferin molecules reach their excited state as a result of oxidative-chemical reactions. Typically, an enzyme from a class of oxidative-enzymes called Luciferase couples with the corresponding Luciferin molecule to catalyse the reaction. In summary, chemiluminescence is the emission of light due to chemical reactions while bioluminescence is the emission of light from living organisms (Mirasoli et al. 2014; Roggo and van der Meer 2017).
Experimental models and measurements to study cardiovascular physiology
Neil Herring, David J. Paterson in Levick's Introduction to Cardiovascular Physiology, 2018
Imaging is a key method of studying cardiovascular physiology and can make use of echocardiography, X-ray computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and single-photon emission CT (SPECT), as described in Chapter 2. A new molecular imaging technique enables the expression of tagged proteins to be imaged in vivo. Bioluminescence (or chemiluminescence) imaging makes use of luciferase, an enzyme that produces light emission from the conversion of D-luciferin to oxyluciferin. D-luciferin can be administered via an intravenous or intraperitoneal injection; then, the animal is anaesthetized to prevent movement artefact, and placed in a dark chamber before imaging with a charge-coupled device camera. The technique is cost-effective and has a high signal-to-noise ratio given the lack of background bioluminescence from mammalian cells. However, it requires genetically encoded luciferase, the injection of the substrate to enable light emission, and the resulting light signal will depend on the depth of tissue being imaged.
Suitability of transiently expressed antibodies for clinical studies: product quality consistency at different production scales
Published in mAbs, 2022
Sara Rodriguez-Conde, Sophie Inman, Viv Lindo, Leanne Amery, Alison Tang, Uche Okorji-Obike, Wenjuan Du, Berend-Jan Bosch, Paul J. Wichgers Schreur, Jeroen Kortekaas, Isabel Sola, Luis Enjuanes, Laura Kerry, Katharina Mahal, Martyn Hulley, Olalekan Daramola
The production of MERS-CoV spike pseudotyped vesicular stomatitis virus (VSV) and neutralization test was performed as described by Widjaja et al.20 HEK 293 T cells (~75% confluency) were transfected with the pCAGGS expression vector encoding the MERS-CoV spike protein with a C-terminal cytoplasmic tail truncation to increase cell surface expression levels. Forty-eight hours post-transfection, cells were infected with VSV-G-pseudotyped VSVΔG bearing the firefly (Photinus pyralis) luciferase reporter gene at a multiplicity of infection (MOI) of 1. Twenty-four hours later, supernatants were harvested and filtered through a 0.45 μm membrane. In the VNT, serially diluted mAbs were pre-incubated with an equal volume of virus at RT for 1 h, after which the mixture was added to Vero-CCL81 cells, and further incubated at 37°C. After 20 h, cells were washed once with phosphate-buffered saline (PBS) and lysed with cell lysis buffer (Promega). The expression of firefly luciferase was measured on a Berthold Centro LB 960 plate luminometer using D-luciferin as a substrate (Promega). The percentage of infectivity was calculated as the ratio of luciferase readout in the presence of mAbs normalized to luciferase readout in the absence of mAb. The half maximal inhibitory concentrations (IC50) were determined using 4-parameter logistic regression (GraphPad Prism v7.0).
Mitochondria autophagy: a potential target for cancer therapy
Published in Journal of Drug Targeting, 2021
Yu-Han Qiu, Tian-Shu Zhang, Xiao-Wei Wang, Meng-yan Wang, Wen-Xia Zhao, Hui-Min Zhou, Cong-Hui Zhang, Mei-Lian Cai, Xiao-Fang Chen, Wu-Li Zhao, Rong-Guang Shao
As one of the metabolic centres of cells, mitochondria produce a large amount of ATP to provide energy for cells. ATP is an important ‘energy currency’ of cells, and plays an important role in various physiological and pathological processes of cells. Changes in the level of ATP will affect the function of cells. Usually a decrease in ATP levels indicates an impairment or decline in the function of the mitochondria. In mitochondria, the decrease of ATP level and the ratio of ATP to ADP may lead to mitochondrial stress and decrease of mitochondrial membrane potential, which in turn leads to the occurrence of mitophagy. Therefore, the detection of mitochondrial ATP level can be used as one of the detection methods of mitophagy. Bioluminescence technology could detect ATP level. The principle of ATP bioluminescence technology is that luciferase uses luciferin, adenosine triphosphate (ATP) and O2 as substrates, and in the presence of Mg2+, it can convert chemical energy into light energy. In the luminescence reaction catalysed by luciferase, ATP has a linear relationship with the luminescence intensity within a certain concentration range [30]. Therefore, the concentration of ATP can be detected with high sensitivity.
Evaluation of wound healing potential of new composite liposomal films containing coenzyme Q10 and d -panthenyl triacetate as combinational treatment
Published in Pharmaceutical Development and Technology, 2021
Ali Asram Sağıroğlu, Burak Çelik, Eray Metin Güler, Abdurrahim Koçyiğit, Özgen Özer
Cell Titer-Glo® Luminescent Cell Viability Test Kit (Promega, Madison, WI, USA) was used to measure the cell viability level. This method determines the degree of cell viability in proportion to the amount of ATP. For analysis, Fibroblast and Primary Epidermal Keratinocytes cells (1.5 × 103 cells/well−1) were plated on 96-well white plates. After 24 h, the cells were incubated with different concentrations of liposomal film containing CoQ10 and PTA (10 to 400 µM for CoQ10 and 25 to 1200 µM for PTA). After incubation, the luciferin derivative and cell lysis solution were added as substrates. The luciferin derivative converts a light signal proportional to the current amount of ATP. Luminescence was measured using a Varioskan Flash Multimode Reader (Thermo Scientific, USA) and normalized to control.
Related Knowledge Centers
- Chemical Compound
- Enzyme
- Oxygen
- Luciferase
- Photoprotein
- Substrate
- Firefly Luciferin
- Firefly Luciferase
- Enzyme Commission Number
- Adenosine Triphosphate