Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture

2012 ◽  
Vol 48 (8) ◽  
pp. 463-468 ◽  
Author(s):  
Aastha Chhabra ◽  
Astha Jaiswal ◽  
Umang Malhotra ◽  
Shrey Kohli ◽  
Vibha Rani
Keyword(s):  
Cell Culture ◽  
Enzyme Activity ◽  
In Situ Zymography

scholarly journals A portable cell culture system with a simple microscope, oxygen sensor and MEA amplifier

2019 ◽  
Author(s):  
Dhanesh Kattipparambil Rajan
Keyword(s):  
Cell Culture ◽  
Chemical Sensing ◽  
Cell Imaging ◽  
Modular System ◽  
Disease Models ◽  
Environment Control ◽  
In Situ Imaging

Cell culture in-vitro is a well-known method to develop cell and disease models for studying physiologically relevant mechanisms and responses for various applications in life sciences. Conventional methods for instance, using static culture flasks or well plates, have limitations, as these cannot provide accurate tractable models for advanced studies. However, microscale systems can overcome this since they mimic the cells' natural microenvironment adequately. We have developed a portable live-cell imaging system with an invert-upright-convertible architecture and a mini-bioreactor for long-term simultaneous cell imaging and analysis, chemical sensing and electrophysiological recording. Our system integrates biocompatible cell-friendly materials with modular measurement schemes and precise environment control and can be automated. High quality time-lapse cell imaging is hugely useful in cell/disease models. However, integration of advanced in-vitro systems into benchtop microscopes for in-situ imaging is tricky and challenging. This is especially true with device based biological systems, such as lab/organ/body-on-chips, or mini-bioreactors/microfluidic systems. They face issues ranging from optical and physical geometry incompatibilities to difficulties in connectivity of flow and perfusion systems. However, the novel modular system we have developed either as an inverted or as an upright system can easily accommodate virtually any in-vitro devices. Furthermore, it can accept additional sensor or measurement devices quite freely. Cell characterization, differentiation, chemical sensing, drug screening, microelectrode-array-electrophysiological recordings, and cell stimulation can be carried out with simultaneous in-situ imaging and analysis. Moreover, our system can be configured to capture images from regions that are otherwise inaccessible by conventional microscopes, for example, cells cultured on physical or biochemical sensor systems. We demonstrate the system for video-based beating analysis of cardiomyocytes, cell orientation analysis on nanocellulose, and simultaneous long-term in-situ microscopy with pO2 and temperature sensing. The compact microscope as such is comparable to standard phase-contrast-microscopes without any detectable aberrations and is useful practically for any in-situ microscopy demands.

scholarly journals Aktivitas NADP(H) Oksidoreduktase pada Kultur Sel Kina (Cinchona ledgeriana Moens) Terelisitasi

2020 ◽  
Vol 25 (4) ◽  
pp. 540-546
Author(s):  
Maulidiyah Utami ◽  
Diah Ratnadewi ◽  
Dyah Iswantini ◽  
Trivadila Trivadila
Keyword(s):  
Cell Culture ◽  
Enzyme Activity ◽  
Secondary Metabolite ◽  
High Performance ◽  
Industrial Plant ◽  
Quinoline Alkaloids ◽  
Quinoline Alkaloid ◽  
Potential Alternative ◽  
Cinchona Ledgeriana

Cinchona ledgeriana Moens is an industrial plant producing secondary metabolite quinoline alkaloids. To maintain and moreover, to increase the quinoline production especially quinine, in vitro culture system through cell culture could be a potential alternative. If the use of elicitor in cell culture can increase the production of a secondary metabolite, the activity of the enzymes involved in the biosynthetic pathway of the secondary metabolite in question might be increasing. This study aimed to examine the activity of NADPH oxidoreductase in the elicitated cell culture of C. ledgeriana and to evaluate the correlation between the activity of this enzyme and the level of quinine production. The cell cultures of Cinchona were treated with abscisic acid (ABA) or paclobutrazol (PBZ), combined with sucrose, sorbitol, or mannitol in Wood Plant (WP) media, for 7 weeks on a shaker. The quinine concentration was determined using high-performance liquid chromatography (HPLC) and the enzyme activity was measured using fluorometry. The results showed that the highest enzyme activity was found in the P7M cells (PBZ 7 mg/L + mannitol 5.3 g/L + sucrose 20 g/L), followed by the A3S cells (ABA 3 mg/L + sorbitol 5.3 g/L + sucrose 20 g/L). These results correspond to their production level of the quinine alkaloids. The lowest enzyme activity was found in the cultures without elicitor. The increase of NADP(H) enzyme activity in the P7M and A3S treatments were 13.5 and 8.5%, respectively, compared to that in the control cells. Keywords: elicitation, fluorometry, NADP(H) oxidoreductase, quinoline alkaloid

Partner telomeres during anaphase in crane-fly spermatocytes are connected by an elastic tether that exerts a backward force and resists poleward motion

2002 ◽  
Vol 115 (7) ◽  
pp. 1541-1549 ◽  
Author(s):  
James R. LaFountain ◽  
Richard W. Cole ◽  
Conly L. Rieder
Keyword(s):  
Cell Culture ◽  
Strong Evidence ◽  
Laser Microbeam ◽  
In Vitro Cell Culture ◽  
Pulling Forces ◽  
As If

As chromosomes move polewards during anaphase in crane-fly spermatocytes,trailing arms commonly stretch backwards for a brief time, as if tethered to their partners. To test that notion, a laser microbeam was used to sever trailing arms and thereby release telomere-containing arm segments (called acentric fragments because they lack kinetochores) from segregating chromosomes. Analysis of the movement of acentric fragments after their release provided clear evidence that previously conjoined partners were indeed tethered at their telomeres and that tethers exerted backward forces that were sufficient to move the fragment across the equator and into the opposite half-spindle. To address concerns that tethers might be artifacts of in vitro cell culture, spermatocytes were fixed in situ, and stretched arms within fixed cells provided strong evidence for tethers in vivo. The substantial resistance that tethers impose on the poleward movement of chromosomes must normally be over-ridden by the poleward `pulling' forces exerted at kinetochores. In spermatocytes, poleward forces are supplied primarily by the`traction fibers' that are firmly attached to kinetochores through end-on attachments to the plus ends of kinetochore microtubules.

In Vitro Pulmonary Cell Culture in Pharmaceutical Inhalation Aerosol Delivery: 2-D, 3-D, and In Situ Bioimpactor Models

2016 ◽  
Vol 22 (17) ◽  
pp. 2522-2531 ◽  
Author(s):  
Maria F. Acosta ◽  
Priya Muralidharan ◽  
Samantha A. Meenach ◽  
Don Hayes ◽  
Stephen M.- Black ◽  
...  
Keyword(s):  
Cell Culture ◽  
Aerosol Delivery

scholarly journals Analysis of glucocerebrosidase activity using N-(1-[14C]hexanoyl)-d-erythro-glucosylsphingosine demonstrates a correlation between levels of residual enzyme activity and the type of Gaucher disease

1994 ◽  
Vol 303 (2) ◽  
pp. 377-382 ◽  
Author(s):  
I Meivar-Levy ◽  
M Horowitz ◽  
A H Futerman
Keyword(s):  
Enzyme Activity ◽  
Lysosomal Storage Disease ◽  
Gaucher Disease ◽  
Storage Disease ◽  
Acyl Chain ◽  
Clinical Severity ◽  
Residual Enzyme Activity ◽  
Lysosomal Storage

Glucosylceramide, a degradation product of complex glycosphingolipids, is hydrolysed in lysosomes by glucocerebrosidase (GlcCerase). Mutations in the human GlcCerase gene cause a reduction in GlcCerase activity and accumulation of glucosylceramide, which results in the onset of Gaucher disease, the most common lysosomal storage disease. Significant clinical heterogeneity is observed in Gaucher disease, with three main types known, but no clear correlation has been reported between the different types and levels of residual GlcCerase activity. We now demonstrate that a correlation exists by using a radioactive, short-acyl chain substrate, N-(1-[14C]hexanoyl)-D-erythro-glucosylsphingosine ([14C]hexanoyl-GlcCer). This substrate rapidly transferred into biological membranes in the absence of detergent [Futerman and Pagano (1991) Biochem. J. 280, 295-302] and was hydrolyzed to N-(1-[14C]hexanoyl)-D-erythro-sphingosine ([14C]hexanoyl-Cer) both in vitro and in situ, with an acid pH optimum. A strict correlation was observed between levels of [14C]hexanoyl-GlcCer hydrolysis and Gaucher type in human skin fibroblasts. The mean residual activity measured in vitro for 3 h incubation in type 1 Gaucher fibroblasts (the mild form of the disease) was 46.3 +/- 4.6 nmol of [14C]hexanoyl-Cer formed per mg protein (n = 9), and in type 2 and 3 fibroblasts (the neuronopathic forms of the disease) was 19.6 +/- 6.5 (n = 9). A similar correlation was observed when activity was measured in situ, suggesting that the clinical severity of a lysosomal storage disease is related to levels of residual enzyme activity.

In Situ and In Vitro Identification and Characterization of Cardiac Ganglion Neurons in the Crab, Carcinus maenas

1999 ◽  
Vol 81 (6) ◽  
pp. 2964-2976 ◽  
Author(s):  
Michelle A. Saver ◽  
Jerrel L. Wilkens ◽  
Naweed I. Syed
Keyword(s):  
Cell Culture ◽  
Carcinus Maenas ◽  
Membrane Properties ◽  
Cardiac Ganglion ◽  
Shore Crabs ◽  
Ganglion Neurons ◽  
Identification And Characterization

In situ and in vitro identification and characterization of cardiac ganglion neurons in the crab, Carcinus maenas. The aim of this study was to investigate the intrinsic membrane properties and hormonal responses of individual central pattern generating neurons in the cardiac ganglion of the shore crab Carcinus maenas. Because the cardiac ganglion in this crustacean species is buried within the heart musculature and is therefore inaccessible for direct morphological and electrophysiological analysis, we developed two novel in vitro preparations. First, to make the ganglion accessible, we established a brief enzymatic treatment procedure that enabled us to isolate the entire cardiac ganglion, in the absence of muscle tissue. Second, a cell culture procedure was developed to isolate individual neurons in vitro. With the use of both isolated ganglionic and neuronal cell culture techniques, this study provides the first direct account of the neuroanatomy of the cardiac ganglion in shore crabs. We demonstrate that cultured neurons not only survived the isolation procedures, but that they also maintained their intrinsic membrane and transmitter response properties, similar to those seen in the intact ganglion. Specifically, we tested the peptides proctolin, crustacean cardioactive peptide, the FLRFamide-related peptide F2, and an amine (serotonin) on both isolated ganglion and in vitro culture neurons. We measured changes in neuronal burst rate, burst amplitude, pacemaker slope, and membrane potential oscillation amplitude in response to the above four hormones. Each hormone either increased neuronal activity in spontaneously bursting neurons, or induced a bursting pattern in quiescent cells. The in vitro cell culture system developed here now provides us with an excellent opportunity to elucidate cellular, synaptic and hormonal mechanisms by which cardiac activity is generated in shore crabs.

IGF-1 loaded injectable microspheres for potential repair of the infarcted myocardium

2020 ◽  
pp. 088532822094850
Author(s):  
Elisabetta Rosellini ◽  
Niccoletta Barbani ◽  
Caterina Frati ◽  
Denise Madeddu ◽  
Diana Massai ◽  
...  
Keyword(s):  
Cell Culture ◽  
Growth Factor ◽  
In Situ Polymerization ◽  
Chemical Imaging ◽  
Homogeneous Distribution ◽  
Insulin Like Growth Factor ◽  
Gelatin Microspheres

The use of injectable scaffolds to repair the infarcted heart is receiving great interest. Thermosensitive polymers, in situ polymerization, in situ cross-linking, and self-assembling peptides are the most investigated approaches to obtain injectability. Aim of the present work was the preparation and characterization of a novel bioactive scaffold, in form of injectable microspheres, for cardiac repair. Gellan/gelatin microspheres were prepared by a water-in-oil emulsion and loaded by adsorption with Insulin-like growth factor 1 to promote tissue regeneration. Obtained microspheres underwent morphological, physicochemical and biological characterization, including cell culture tests in static and dynamic conditions and in vivo tests. Morphological analysis of the microspheres showed a spherical shape, a microporous surface and an average diameter of 66 ± 17µm (under dry conditions) and 123 ± 24 µm (under wet conditions). Chemical Imaging analysis pointed out a homogeneous distribution of gellan, gelatin and Insulin-like growth factor-1 within the microsphere matrix. In vitro cell culture tests showed that the microspheres promoted rat cardiac progenitor cells adhesion, and cluster formation. After dynamic suspension culture within an impeller-free bioreactor, cells still adhered to microspheres, spreading their cytoplasm over microsphere surface. Intramyocardial administration of microspheres in a cryoinjury rat model attenuated chamber dilatation, myocardial damage and fibrosis and improved cell homing. Overall, the findings of this study confirm that the produced microspheres display morphological, physicochemical, functional and biological properties potentially adequate for future applications as injectable scaffold for cardiac tissue engineering.

scholarly journals Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3552
Author(s):  
Kristina Perminaite ◽  
Mindaugas Marksa ◽  
Monika Stančiauskaitė ◽  
Tadas Juknius ◽  
Aidas Grigonis ◽  
...  
Keyword(s):  
Cell Culture ◽  
Refractive Index ◽  
Cell Viability ◽  
Royal Jelly ◽  
Biological Activities ◽  
Pharmaceutical Formulations ◽  
Exposure Test ◽  
Cell Culture Line

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.

scholarly journals Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

2009 ◽  
Vol 58 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Elin Hadler-Olsen ◽  
Premasany Kanapathippillai ◽  
Eli Berg ◽  
Gunbjørg Svineng ◽  
Jan-Olof Winberg ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
In Situ Zymography
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