Principle and applications of glucose uptake assay and calcium influx assay
Diabetes Mellitus and its types
Calcium regulation
Glucose regulation and how it is released in cells.
Assignment on Preclinical Screening of ImmunomodulatorsDeepak Kumar
Assignment on Preclinical screening of new substances for the pharmacological activity using in vivo, in vitro, and other possible animal alternative models
Introduction to Screening Models Of Anti Cancer Drugs
Need for novel anti cancer drugs, In - vitro methods, In - vivo methods, Advantages and disadvantages
Presented by
T. Niranjan Reddy
Department of Pharmacology
Assignment on Preclinical Screening of ImmunomodulatorsDeepak Kumar
Assignment on Preclinical screening of new substances for the pharmacological activity using in vivo, in vitro, and other possible animal alternative models
Introduction to Screening Models Of Anti Cancer Drugs
Need for novel anti cancer drugs, In - vitro methods, In - vivo methods, Advantages and disadvantages
Presented by
T. Niranjan Reddy
Department of Pharmacology
Introduction to Screening Models of Anti-Atherosclerosis
Atherosclerosis, Screening models, In vitro models, In vivo models
Presented by
SHAIK FIRDOUS BANU
Department of Pharmacology
Pancreatic hormone - Endocrinology for biochemistryASHA SIVAJI
Pancreatic hormone - In this you will know about synthesis, metabolism, mode of action, biological actions, regulation and disorders related with insulin,Glucagon, Pancreatic somatostatin and pancreatic polypeptide.
Introduction to Screening Models of Anti-Atherosclerosis
Atherosclerosis, Screening models, In vitro models, In vivo models
Presented by
SHAIK FIRDOUS BANU
Department of Pharmacology
Pancreatic hormone - Endocrinology for biochemistryASHA SIVAJI
Pancreatic hormone - In this you will know about synthesis, metabolism, mode of action, biological actions, regulation and disorders related with insulin,Glucagon, Pancreatic somatostatin and pancreatic polypeptide.
Reversible Cryopreservation of Living Cells Using an Electron Microscopy Cryo...AnimatedWorld
Cryo-preservation is a process where organelles, cells, tissues, extracellular matrix, organs, or any other biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures (typically −80 °C using solid carbon dioxide or −196 °C using liquid nitrogen)
Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy and cryo-electron microscopy.
Rapid cooling of aqueous solutions is a powerful tool in life science for at least two important biological and biomedical applications:
(I) cryofixation of samples for (ultra-) structural investigations by (cryo-) microscopy,
(II) cryopreservation of living samples for long-time storage.
Cells undergoing morphological changes during apoptosisAnimatedWorld
Cell culture- Chicken DU249 Hepatoma cell grown in RPMI 1640 plus 10% FBS in Monolayer or suspension culture preparation of S/M Extract.
Microscopy- Fluorescence microscopy was performed using an Olympus Vanox microscope equipped with a filter set from chromo technology corporation.
Images were acquired using a DAGE SIT camera controlled by Hyperscope image analysis program.
Staining Technique - MPM-2STAINING
Murine thymocyte treated with 0.1uM dexamethasone were attached to Adhesio-Slides . SN12C cells were grown on coverslip.
Cells were fixed 4% formaldehyde processed for indrec immunofluoresence using MPM-2 antibody diluted 1:200 and biotinylated horse anti-mouse secondary antibody with streptavidin-texas red.
overexpression of mrps18 2 in cancer cell lines resultsAnimatedWorld
Human mitochondrial ribosomal protein MRPS18-2 (S18-2) is encoded by a cellular gene that is located on the human chromosome 6p21.3.
They observed the overexpression of the S18-2 protein led to immortalization and de-differentiation of primary rat embryonic fibroblasts and then Cells showed anchorage-independent growth pattern.
There observation is not limited to overexpression but they also see the evidence of s18-2 somehow involves in cell cycle regulation and cause disturbances.
S18-2 protein when over expressed it also cause appearance of multinucleated cells in the selected clones.
Regulatory guidelines for conducting toxicity studies by ichAnimatedWorld
ICH is the “International Conference on Harmonization of
Technical Requirements for Registration of Pharmaceuticals for
Human Use”
ICH is a joint initiative involving both regulators and research based industry representatives of the EU, Japan and the US in
scientific and technical discussions of the testing procedures required
to assess and ensure the safety, quality and efficacy of medicines
Anesthesia and euthanasia of experimental animal by vivek and naveenAnimatedWorld
Anesthesia and euthanasia of experimental animal by vivek and naveen
Anesthesia
It is a state of controlled temporary loss of sensation or awareness that or awareness that is induced for medical purpose.
Anesthetic agents
The anesthetic agents are great and choosing the correct one for particular suggestion.
In laboratory animal field , the anesthetic surgeon and post operative are often one and the same person.
This will help to chose correct drug for anaesthesia.
Sometime the wise anesthetic agents also cause undesirable responses. so, its responsibility of experimenters to document this advance in exprimental protocol
Euthanasia
The term euthanasia is derived from the Greek terms eu mean good and thanatos mean death.
Euthanasia is the act of including humane death in an animal. sacrificing the experimental animal after use by gentle procedure causing minimum of physical and mental suffering is called euthanasia.
Classification of receptors family by vivek sharmaAnimatedWorld
Definition- Receptor are the biologic molecule to which drug bind and produces a measurable response.
So, enzyme and structural proteins can be considerd to be pharmacologic receptors.
Majorly receptor are of 4 types and the molecule or a drug interact to receptor to give response often called as ligand.
The type of receptor a ligand will bind is depend on the nature of ligand.
Hydrophilliic ligand binds to the receptor found on the cell surface.
Hydrophobic ligand can enter the cell membrane to intract the receptor present on inside the cells.
Classification of Receptors
A. Cell surface receptor
Ligand-gated Ion Channel
G Protein Coupled Receptor
Enzyme linked Receptor
B. Intracellular Receptor
Nuclear Receptor
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
3. Introduction
Glucose is the primary source of energy for many
organisms, and the uptake of glucose is a critical
process. Glucose is transported across the cell’s
membrane and trapped by being phosphorylated.
In mammalian cells, this is performed by a family of
glucose transporters (GLUT) and a few intracellular
hexose kinases.
measuring glucose uptake is not the same as measuring
glucose consumption.
Glucose uptake occurs on a rapid time scale of 10
minutes or less and specifically measures transporter
activity, whereas changes in glucose concentration
involve a multitude of pathways and typically take
several hours.
4. Diabetes mellitus:
Diabetes mellitus (DM) is a group of diseases characterized by high
levels of blood glucose resulting from defects in insulin production,
insulin action, or both.
The effects of diabetes mellitus include long–term damage,
dysfunction and failure of various organs.
7. TYPESOF DM
Type 1 DM
It is due to insulin deficiency and is formerly known as.
Type I
Insulin Dependent DM (IDDM)
Juvenile onset DM
It is due to pancreatic islet β-cell destruction
predominantly by an autoimmune process.
Usually develops in childhood or early adulthood
It accounts for upto 10% of all DM cases
Develops as a result of the exposure of a genetically
susceptible individual to an environmental agent
8. Type 2 DM:
It is a combined insulin resistance and relative deficiency in insulin secretion and
is frequently known as.
Type II
Noninsulin Dependent DM (NIDDM)
Adult onset DM
It results from insulin resistance with a defect in compensatory insulin secretion.
Insulin may be low, normal or high!
About 30% of the Type 2 DM patients are undiagnosed (they do not know that
they have the disease) because symptoms are mild.
It accounts for up to 90% of all DM cases
9. PRINCIPLE
Glucose uptake activity was analyzed by measuring the rate of
uptake of radioactively tagged 2-deoxy glucose in differentiated
3T3 L1 cells.
• After starvation the cells were treated with insulin and other plant
extracts. Then these ligands will bind to the receptors on the surface
of the cells.
• These triggered the translocation of glucose transporters to the cell
surface.
• Then we treat it with radioactive cocktail containing10µM 2-
deoxy glucose and 0.25µCi of 2 deoxy-D-(3H) – glucose. So the
radioactively tagged glucose will enter the cell along with the
normal glucose.
• By measuring this uptake rate by using liquid scintillation counter
help to analyze the glucose uptake activity and the effect of plant
extract on the glucose uptake activity
10. Why Measure
Glucose
Uptake
Changes in glucose uptake can reflect overall changes in
metabolism, but there are many specific processes as well.
In cancer cells, measuring glucose uptake can monitor the over
expression of glucose transporters or identify glucose transporter
inhibitors.
With fat and muscle cells, changes in GLUT4 translocation upon
insulin stimulation can be observed by measuring glucose uptake.
In immunologically relevant cells, measuring glucose uptake can
be used to follow the transformation of certain cell types from one
stage to another.
11.
12. Glucose
uptake
assay
Using
3T3
L1 cells
• Insulin promotes glucose uptake, metabolism and
storage in adipose tissue and skeletal muscle.
• Insulin stimulates phosphorylation of insulin receptor
substrates (IRS) by kinase, which leads to activation of
PI3 kinase, PKB and protein kinase C isoforms.
• Activated PKB translocate Glut4 to the cell surface
and stimulate glucose transport in muscle and fat cells,
whereas it phosphorylates and inhibits GSK-3.
Inhibitor of GSK-3 enhances insulin signalling thereby
favouring glucose entry into the muscle cells and
adipose tissue.
• Inactivation of GSK-3 in 3T3 L1 differentiated cells
[adipocytes] stimulates glucose uptake which can be
measured by incorporation of 2-deoxy- 14C-glucose
and quantified by using scintillation counter.
13. Cell culture
•The cell line 3T3-L1 pre- adipocytes are cultured and made to
differentiate.
• The pre- adipocytes are then treated with 0.5 mM 3- isobutyl
methylxanthine, 5 μg/mL insulin and 0.25 μM dexamethasone in
10% FBS containing DMEM for 2 days. Later, the cells are
transferred to 10% FBS/DMEM containing only insulin and then to
10% FBS/DMEM without insulin for next 2 days.
• After ten days of differentiation, the cells are used for the
experiments. Chinese hamster ovaric cells over expressing the
human insulin receptor (CHO-IR) cells are grown in Ham's F12
supplemented with 100 U/mL penicillin; 10% fatal bovine serum;
2.5 μg/mL fungizone; 0.5% G-418 and 100 mg/ml streptomycin in
5% CO2/humidified atmosphere at 37°C. Cells are passed two to
three times a week.
• Confluent cells are used for the experiments.
14. Glucose
uptake
assay using
3T3- L1
adipocytes
• 2-deoxy-D-[3H] glucose uptake of 3T3-L1 adipocyte is used to
measure the glucose transport system38.
• 3T3-L1adipocyte cells cultured on 12 well microtitre plate are
incubated in a transport solution containing 1 μCi 2- deoxy D-[3H]
glucose (10 mCi/ mmol) and 0.1 mM 2- deoxy-Dglucose for 7
minutes.
• Uptake of glucoseis terminated by the addition of 50 mM glucose
and 0.1MNaOH/ 0.1% PBS for disruption of cells.
• Radioactivity incorporated in the cells is determined using
scintillation counter.
• Protein is used to standardize the glucose transport values
15. Glucose
uptake
by isolated
diaphragm
from
mice &rat
To study the effect of Insulin & Insulin like substance on muscle
tissue
To study glycogen synthesis.
To study glucose transport.
To study glycogen synthase activity.
16. Count.
Glucose uptake activity was analyzed by measuring the rate of
uptake of radioactively tagged 2-deoxy glucose in differentiated
3T3 L1 cells.
• After starvation the cells were treated with insulin and other
plant extracts.
•Then these ligands will bind to the receptors on the surface of
the cells.These triggered the translocation of glucose transporters
to the cell surface.
Then we treat it with radioactive cocktail containing10µM 2-deoxy
glucose and 0.25µCi of 2 deoxy-D-(3H) – glucose.
• So the radioactively tagged glucose will enter the cell along with
the normal glucose.
• By measuring this uptake rate by using liquid scintillation
counter help to analyze the glucose uptake activity and the effect
of plant extract on the glucose uptake activity.
17. Application of
glucose
uptake assay
Glucose uptake experiments are commonly used to measure cellular
metabolic activity and glucose transport. Glucose uptake can be
studied using radiolabeled glucose itself, or radiolabeled glucose
analogs such as 2-deoxy-D-glucose (DOG) or 3-O-methyl-D-
glucose .
18. Introduction
CALCIUM
INFLUX
ASSAY
Based on second messenger assay which is used to measure the
calcium flux associatedGq-protein coupled receptor (inhibition or
activation).
Done by using calcium sensitive fluorescent dye and is mostly
taken up into cytoplasm.
For anion transporter cells probencid (inhibitor of anion
transporter) used for retention of florescent dye in cell
Dye binds to the released calcium and fluorescence intensity
increases.
Change in fluorescence intensity directly proportional to amount
of calcium released.
19. CALCIUM
INDICATORS
Types Of Dye
I. Luminescent Dye:- eg. Aequrin and obelin
II. Fluorescent Dye :- eg.Calcein8,
Chlortetracycline,Fluo-3,Fluo-8,Rhod-4
III. Calcium Senstive Dye:-eg. Tetra-
methylmurexide TM,arsenazo IIP,anti-
pyrylazo III and chlorphophonazo LiP.
20. Detection
techniques
I. Flow Cytometric Analysis:
II. ColorimetricAssay
III. Luminometric Assay
IV. Fluorometric Assay
V. Fluo-8Calcium Assay
VI. Rhod 4 calciumAssay
VII. Cal-520,AM Assay
VIII. Ratiometric Analysis
21. i) Flow
Cytometric
Analysis
Flow cytometry (FC) is a technique used to detect and
measure physical and chemical characteristics of a population
of cells or particles.
• Phorbol myristate acetate and
calcium ioophoe ionomocinStimulant
• T-Cells, RPMI media, Fetal
bovine Serum, peniccilin
Other Material
and reagent
• Centrifuge,37 C 5% CO2 Cell
Culture incubator,Cell counterEquipment
• Flow Jo Software
Software
23. Procedure
T- Cell preparation
Preparation of Indicator and Enhencing solution
Loading dye preparation by mixing indicator and enhancing solution
Loading dye into cells and incubate for 1 hour in 37C CO2 incubator
Cool down the tube 20 minutes before analysis.
24. Cont.
ATP dose dependent (10µM,1µM or 0µM ATP) Intracellular calcium
measured by flow cytometry
Cells were incubated with 520AM dye for 30 min at 37C before ATP was
Added into the cells.
The baseline was acquired and the rest of cells were analyzed after ATP
addition
25. II)Luminometric
Assay
More sensitive then fluorescence based calcium assay
Provides an optimized assay method for monitoring the
GPC and calcium channels
Useful for monitoring the intracellular calcium
mobilization in a specified compartment given that
recombinant apoaequorin proteins.
More sensitive than the fluorescent calcium assays
26. Principle
and
Procedure
Luminometric assay are preferred methods in drug discovery for screening G protein coupled receptors (GPCR).
It uses a highly calcium- sensitive and membrane- permeable coelenterazine-calcium indicator for the cells that are transfected
with apoaequorin gene.
Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea victoria that has been used extensively as a
calcium indicator in cells
The aequorin complex emits blue light when bound to calcium ions.The luminescence intensity is directly proportional to the Ca2+
concentration. 23
Cells transfected with apoaequorin .The growth medium was removed and the cells were incubated with 100 µL of dye- loading
solution using for 3 hours at RT and protected from light. ATP (25 µL/well) was added to achieve the final indicated concentrations.
The EC50 of ATP is about 0.8 µM.
27. III) Fluo-8
Calcium Assay
It is a fluorescence-based assay for detecting intracellular
calcium mobilization.
• It provides an optimized assay method for monitoring the
G-protein-coupled receptors and calcium channels
Principle:- Cells expressing a GPCR of interest that signals
through calcium are pre-loaded with Fluo-8 which can cross the
cell membrane.
Once inside the cell, the lipophilic blocking groups of Fluo-8 are
cleaved by an esterase, resulting in a negatively charged
fluorescent dye that stays inside the cell.
28. Cont.
Its fluorescence is greatly enhanced upon binding to calcium.
When cells are stimulated with agonists, the receptor signals
the release of intracellular calcium, which significantly
increases the fluorescence of Fluo-8.
Cells incubated with 100 µL of dye loading solution using
Fluo-4 and Fluo-8 for 1 hour at room temperature. Carbachol
(50 µL/well) was added to achieve the final concentrations
indicated.
29. IV)Colorimetric
assay
colorimetric calcium assays use reagents that
measurable color change in the presence of an
analyte .
Example :o-cresolphthalein reacts with calcium
in an alkaline environment to produce a violet
colored complex. And color intensity is
proportional to calcium concentration in the
sample .
Colorimetric assay is the quick and easy assay
can be used to determined the calcium
concentration.(physiological range 0.4-
110mg/dl.