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What is Immunocytochemistry? Immunocytochemistry Methods, Techniques
Continue ReadingAbout Immunocytochemistry
Immunocytochemistry (ICC) is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes.
The antibodies bound on diffirent epitotoes can then be detected using several different methods.
Immunocytochemistry enables researchers to evaluate whether or not cells in a particular sample express the antigen in question.
In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.
In the present experiment we are identifying the neurons by the presence of neuron-specific marker Neurofilament-H in the primary cotical neuronal culture.
Adopted from BioRender
Immunocytochemistry Requirements
1. 4% Para formaldehyde in PBS
2. Triton X-100, Milli Q water
3. Bovine serum albumin (BSA)
4. 5% w/v BSA in Phosphate buffered saline (PBS)
5. 0.1% w/v BSA in PBS
6. Primary antibody produced in rabbit
7. Goat-Anti rabbit secondary antibodies.
8. Inverted florescent microscope
9. TMB substrate for localization(20X)
Procedure of Immunocytochemistry
1. Wash the cultures two times with PBS and then fix for 30 minutes with 4% paraformaldehyde – PBS at room temperature followed by permeabilisation with 0.2% Triton X-100 for 5 minutes.
2. Block the non-specific binding by incubating the cells with PBS containing 5% BSA w/v for 1 h at room temperature.
3. After blocking, wash once with PBS and incubate with primary antibody overnight at 4°C In the present experiment we are using specific antibody as neuronal marker.
The antibody should be diluted with PBS with 0.1% BSA (w/v) according to manufacterur’s instructions.
In the present case we are diluting 1part of antibody with 399 parts of 0.1% BSA-PBS.
4. Once the incubation time is over, remove the primary antibody and wash three times with 0.1% BSA-PBS
5. After washing incubate the cultures with secondary antibodies which was goat anti rabbit IgG conjugated with HRP (1:3000) in 0.1% BSA-PBS for 1 hour at room temperature.
6. Following the incubation, remove the antibody solution and wash three more times with 0.1% BSA-PBS.
7. Add 200μl of TMB substrate to each of the well and incubate in dark for few minutes.
Remove the substrate as soon as color starts appearing and observe under the microscope
Immunocytochemistry Citations
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Cytoplasm: Definition, Function, and Examples
Continue ReadingWhat is Cytoplasm?
Cytoplasm is the liquid matrix of the cell in which the membrane bound organelles such as Mitochondria, Golgi Apparatus ribosome, nucleus and other chemical components proteins, Glucose, Lipids.
The cytoplasm was first discovered by Anton Von Leuwenhoek and termed by Kolliker in 19th century.
Though cytoplasm is a liquid component the diverse make of the components provides a structural organization and the components are sequentially arranged and are well synchronized.
The cytoplasm is an indicator to the outside environment and changes the activity of the cell frequently to maintain HOMEOSTASIS.
Characteristic of Cytoplasm
1. Cytoplasm is a well – organized liquid matrix:
Earlier it was considered as a part of protoplasm but invention of Electron Microscopes provided a depth of knowledge regarding the organization of cytoplasm inside the cell.
2. The movement of matrix is well programmed:
Based on the cell’s necessity, transportation of enzymes, proteins and other substances which are required at different part of the cell.
3. Cytoplasm is the internal milieu of the cell:
They act as regulators maintaining a cell’s vital survival function even the external factors disrupt the homeostatic function and maintain the internal environment.
4. Maintains the integrity of the cell:
The cell shape, movement, protein synthesis and other metabolic activities are governed by the cytoplasm.
5. High viscosity matrix and colloid:
The thickness of the matrix is usually higher than that of water. Higher the viscosity easier the organelles can suspend. Because of the suspension of the organelles the matrix is colloid.
Components of Cytoplasm
Cytoplasm has both solid and liquid components of Cytoplasm.
Solid Components are
1. Organelles bound by two membrane
a. Plastids – special feature present only in plant cell has a variety of types and function. The 2 types are Leucoplasts and Chromoplasts.
i. Leucoplasts are colorless components which have their role in storage and metabolism of starch.
ii. Chromoplasts are colored compound mainly green in plants forming the chloroplasts which are essential in photosynthesis.
b. Mitochondria – Power house of the cell provides energy in ATP from the cellular respiration.
2. Organelles Bound by one membrane
a. Peroxisomes – Peroxisomes are protective in function producing peroxides
b. Vacuoles – Specialized feature present in plants where the food molecules are stored for metabolism when needed.
3. Ribosomes – Ribosomes are molecular compounds which is involved in formation of protein for cell structural function.
4. Endomembrane system – consists Endoplasmic Reticulum, Golgi Apparatus and Nuclear Envelope
5. The endomembrane system constitutes the internal membrane providing organelle free matrix for cell survival.
The endoplasmic reticulum arises from the nucleus extends throughout the length and reaches plasma membrane forming the endomembrane.
I. Nuclear envelope: Nuclear Envelope is made up of flattened cisternae like discs. Many discs come together forming a small pore which allows a contact between cytoplasm and nucleus.
The outer membrane contacts endoplasmic reticulum and the inner membrane has had thread of chromatin molecule.
II. Endoplasmic Reticulum (ER) is the major constituent of the endomembrane system. They are tubule and flattened that carries out the function of storage.
The ER is of 2 types:
a. Rough Endoplasmic Reticulum
b. Smooth Endoplasmic Reticulum protein synthesized are stored at RER but transported through SER.
Their main function remains secretion.
III. Golgi Complex flattened stack of disc from dictyosomes forms the determined structure.
Golgi Bodies are concerned with packaging and storing of the secretory molecule and then vesicles are pinched off from Golgi complex through exocytosis.
6. Cytoskeleton
I. Micro tubules – Microtubules are thin and rigid structures made up of 13 filaments. Protein component is the main structural source for the tubule structure is contributed by Tubulin.
They are easily depolymerized and can shape and function of any cell by contributing to Cell division providing spindle fibers, Asters, Locomotion etc.,
II. Centrioles – cylindrical structure and paired. They migrate to animal pole during cell division
III. Basal Bodies – a structure similar to centrioles are present at the bases of cilia or flagella. Cilia and Flagella both has a common arrangement of microtubule (9 + 2). These are used in order for locomotion purposes.
IV. Microfilaments – are smallest structure made up of actin associated with myosin and other aiding proteins which help in contraction thereby causing motility of the organism.
V. Intermediate Filaments – they are mechanical in function
Liquid components are:
1. Cytosol – A liquid component comprises the structural organelles of cell. They are present inside the cell. Responsible for movement and integrity.
2. Hyaloplasm – is the ground cytoplasm which does not have any structural component. They are present outside the endomembrane system.
Their main role is to regulate the internal milieu. This is also called as Ground cytoplasm / Cytoplasmic matrix proper.
Other components Excretory products, Metabolic storage, Secretory materials
Cytoplasm: Plant vs Animal Cells
The difference between plant and animal cell cytoplasm is negligible as the cytoplasm has common role in both cell types.
The main difference will be the presence and absence of organelles present in them such as vacuoles, chloroplasts etc.,
Significance of Cytoplasm
1. Microtubules specifically locate and guide the enzymes. Precise positioning information is provided for the enzymes to catalyst the biosynthesis.
2. The ER is related to the formation of glyoxysomes and vacuoles
3. Golgi Complex is distributed in the cytoplasm in the form of dictyosomes.
4. Cytoplasmic Streaming – Also known as CYCLOSIS, is a process where the cell components are circulated inside the matrix for maintain the process of homeostasis.
The streaming is influenced by the pH, temperature and various other factors which modify the body’s homeostatic condition.
For Example: Sunlight falls on a particular spot of the plant. The other regions lack sunlight here the cytoplasmic streaming with the help of microtubules and filaments the chloroplast is moved to sun receiving regions.
5. The structural integrity, motility, shape are all maintained by the Cytoplasm.
Cytoplasm Citations
- Consequences of phase separation in cytoplasm. Int Rev Cytol . 2000;192:331-43.
- The periplastidal compartment: a naturally minimized eukaryotic cytoplasm. Curr Opin Microbiol . 2014 Dec;22:88-93.
- How crowded is the cytoplasm? Cell . 1982 Sep;30(2):345-7.
- Molecular pathways involved in the transport of nuclear receptors from the nucleus to cytoplasm. J Steroid Biochem Mol Biol . 2018 Apr;178:36-44.
- Cytoplasm’s Got Moves. Dev Cell . 2021 Jan 25;56(2):213-226.
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Glucose Oxidation Respiratory Balance Sheet
Continue ReadingAbout Glucose Oxidation Respiratory Balance Sheet
Respiratory balance sheet is a mathematical calculation of net ATP produced by cellular respiration during glucose oxidation which includes Glycolysis, Krebs Cycle and Oxidative Phosphorylation under aerobic condition utilizing one glucose molecule.
Features of Glucose Oxidation Respiratory Balance Sheet
Certain assumptions were considered while forming the balance sheet:
1. For an oxidizing Glucose molecule, it must follow the cellular respiration process in a sequence such that glucose must follow every step of glycolysis to form 2 molecules of pyruvate.
Pyruvate must enter Krebs Cycle to produce reducing equivalents.
The reducing equivalents from Glycolysis and Krebs cycle must enter Electron Transport Chain where they get converted into ATP.
2. Reducing equivalents such as the NADH must carried to Electron Transport Chain in mitochondria
3. The intermediates formed during the oxidization of glucose should not participate in any biosynthesis process to form other products such as Amino acids, Purines pyrimidines or porphyrins.
4. The cellular respiratory substrate must only be GLUCOSE other substrates must not be considered.
Considering the dynamic nature of our cell and living system the above assumptions do not apply practically for a working cell. The balance sheet assumptions are theoretically accepted and followed.
Glucose Oxidation Respiratory Balance Sheet
When NADH produces 3 ATP and FADH2 produces 2 ATP
Respiration Process Direct Synthesis In ETC ATP Consumed Net Gain NADH+FADH2 Glycolysis 4 6+Nil 2 8 Krebs Cycle 3 18+4 Nil 24 Acetyl CoA Formation Nil 6+Nil Nil 6 Total Gain 6 30+4 -2 38 When NADH produces 2.5 ATP and FADH2 produces 1.5 ATP
Respiration Process Direct Synthesis In ETC ATP Consumed Net Gain NADH+FADH2 Glycolysis 4 5+Nil 2 7 Krebs Cycle 2 15+3 Nil 20 Acetyl CoA Formation Nil 5+Nil Nil 5 Total Gain 6 25+3 -2 32 Under Anaerobic respiration, the pyruvic acid converts to lactic acid producing only 6 ATP.
The list of reactions which produced direct ATP synthesis and reduced equivalents are given below:
Glucose Oxidation: Glycolysis
1. Glyceraldehyde 3 Phosphate + NAD → 1,3 – diphosphoglycerate + NADH
Enzyme: Glyceraldehyde – 3 phosphate dehydrogenases
2. 1,3 diphosphoglycerate +ADP + Pi → 3 – phosphoglycerate + ATP
Enzyme: Phosphoglycerate Kinase
3. Phosphoenol pyruvate + ADP + Pi → Pyruvate + ATP
Enzyme: Pyruvate Kinase
These 3 reactions take place twice therefore they produce 4 ATP + 2 NADPH = 4 + 2 (3) = 10
Two other reaction consumes ATP
1. Glucose + ATP → Glucose 6 phosphate + ADP + Pi
Enzyme: Hexokinase
2. Fructose 6 Phosphate + ATP → Fructose1,6 diphosphate + ADP + Pi
Enzyme: Phosphofructokinase
ATP consumed is 2. Therefore, Glycolysis as a whole provides 8 ATP.
Under Anaerobic condition:
2Pyruvate + 2NAD → 2Lactate/ethanol + 2NADH → 6 ATP
Glucose Oxidation: Citric Acid Cycle
1. 2 Pyruvate + 2 NAD → 2 Acetyl Co – A + 2 NADH
Enzyme: Pyruvate Dehydrogenase
2. 2 Isocitrate + 2 NAD → 2 Oxalosuccinate + 2 NADH
Enzyme: Isocitrate Dehydrogenase
3. 2 α – ketoglutarate + 2 NAD → 2 Succinyl Co – A + 2 NADH
Enzyme: α – ketoglutarate Dehydrogenase
4. 2 Succinyl Co – A + 2GDP + 2Pi → 2 Succinate + 2GTP
Enzyme: Succinate Thiokinase
5. 2 Succinate + 2FAD → 2Fumarate + 2FADH2
Enzyme: Succinate Dehydrogenase
6. 2 Malate + 2 NAD → 2 oxaloacetate + 2 NADH
Enzyme: Malate Dehydrogenase
2 pyruvate molecules undergo complete oxidation and provide 30 ATP molecules.
Glucose Oxidation Citations
- Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr Opin Plant Biol . 2004 Jun;7(3):254-61.
- The effect of selenium-deficiency on rat fat-cell glucose oxidation. Biochem J . 1983 Aug 15;214(2):471-7.
- Ascorbic acid and glucose oxidation by ultraviolet A-generated oxygen free radicals. Invest Ophthalmol Vis Sci . 1996 Jul;37(8):1549-56.
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Cell Viability Assays: Overview of Cell Viability...
Continue ReadingAbout Calcein AM / PI Cell Viability Assays
The Calcein –AM/PI assay is a two-colour fluorescence cell death assay that is based on the simultaneous determination of live and dead cells with two different probes.
It measure recognized parameters of cell viability by evaluating intracellular esterase activity and plasma membrane integrity : calcein-AM and Propidium iodide (PI) .
The assay principles are general and applicable to most eukaryotic cell types, including adherent cells, suspension cells, and certain tissues, but not to bacteria or yeast.
The viable cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell- permeant calcein AM to the intensely fluorescent calcein.
The calcein, a polyanionic dye is well retained within viable cells, producing an intense uniform green fluorescence in viable cells (ex/em ~495 nm/~515 nm).
Propidium iodide enters cells with damaged membranes in dead cells and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids (DNA, RNA) thereby producing a bright red fluorescence in dead cells (ex/em ~535 nm/~617 nm).
Adopted from from BioRender
Cell Viability Assays Principle
PI is excluded by the intact plasma membrane of live cells.
The cell viability determination depends on these physical and biochemical properties of cells.
Cytotoxic events that do not affect these cell properties may not be accurately assessed using this method.
Background fluorescence levels are inherently low with this assay technique because the dyes are virtually non- fluorescent before interacting with cells.
This assay is suitable for use with a wide variety of techniques, including microplate assays immunocytochemistry, flow cytometry, and in vivo cell tracing
Cell Viability Assays Requirements
1. Calcein-AM fluorescent dye
2. Propidium iodide fluorescent dye
3. Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).
4. Fluorescence microscope with blue (FITC) and green (Rhodamine) filters 5. 37oC water bath
Cell Viability Assays Procedure
1. Prepare the stock of Calcein-AM dye in DMSO at a concentration of 1mg/ml. Similarly prepare PI stock in sterile water at a concentration of 10mg/ml
NOTE: These dyes are highly light sensitive and should be stored in smaller aliquots frozen in -20`C till use; PI is a suspected mutagen and hence should be handled with great care
2. Adherent cells may be cultured on sterile glass coverslips as either confluent or subconfluent monolayers (e.g., fibroblasts are typically grown on the coverslip for 2–3 days until acceptable cell densities are obtained).
The cells may be then cultured inside 35 mm disposable petri dishes or other suitable containers; non-adherent cells may also be used.
NOTE: If inverted fluorescence microscope is used, then the cells can be grown in the culture plates and observed directly in the microscope If a normal (upright) fluorescence microscope is available, cells can be grown on coverslips, loaded with dye and carefully mounted on glass slides, and observed under microscope.
3. Remove the medium from the cells in the plates (for cells in suspension, centrifuge and remove the medium; for adherent cells medium can be directly removed).
4. Wash twice with PBS buffer. It is important to wash twice as traces of serum in the medium might interfere with the loading of the dyes.
5. Add fresh buffer and add Calcein-AM dye so that the final concentration of the dye is 1μg/ml.
6. Incubate at 37`C in dark for 15 minutes
7. Now add PI dye so that the final concentration is 10μg/ml. Incubate in dark at 37`C for a further 5 minute duration
8. Now remove the dye containing buffer and add fresh buffer.
9. Observe under the microscope FITC filter can be used for viewing the calcein stained live cells and Rhodamine filter can be used for PI stained dead cells
10. Count the number of calcein positive cells and PI positive cells also the total number of cells in each field of view. Count atleast five fields for each plate.
11. The cell viability can be expressed as (the number of calcein positive cells/ total number of cells) x 100
Features of About Calcein AM / PI Cell Viability Assays
1. Suitable for both proliferating and non-proliferating cells
2. Ideal for both suspension and adherent cells
3. Rapid (no solubilization step)
4. Ideal for high-throughput assays
5. Better retention and brightness compared to other fluorescent compounds (i.e. fluorescein)
6. Useful in a variety of studies, including cell adhesion, chemotaxis, multi-drug resistance
Cell Viability Assays Citations:
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Citric Acid Cycle: an Overview, Mechanism, and,...
Continue ReadingWhat is Citric Acid Cycle?
Citric Acid Cycle is an essential catabolic cycle of cellular respiration involves pyruvate as an end product from glycolysis to form energy in terms of ATP, NADH and FADH2. Which in further processes to provide ATP.
The Citric Acid Cycle takes place in Mitochondria and are coupled with Electron Transport Chain to produce ATP.
Hence Mitochondria is the power house of the cell. The Citric Acid Cycle was discovered by Hans Kreb in 1937 from animal enzymes.
Therefore, it can be named as the Krebs’s Cycle or Tricarboxylic Acid Cycle (Contains 3 carboxyl group in the component).
Citric Acid Cycle Feature
1. Takes place during aerobic respiration:
Pyruvate under aerobic condition produces energy and under anaerobic condition they undergo a process of fermentation forming lactic acid or alcohol
2. TCA Cycle is irreversible at 3 steps: producing high amount of negative energy.
The 3 steps involve the enzymes: citrate synthase, α – ketoglutarate dehydrogenase and isocitrate dehydrogenase.
3. Pyruvate; an end product of glucose metabolism; is transported from cytosol to mitochondria to become Acetyl Co – A.
Pyruvate is transported to mitochondria through Voltage gated channel crossing outer mitochondrial membrane. Mitochondrial Pyruvate Carrier then takes pyruvate through Inner Mitochondrial Membrane.
Pyruvate Dehydrogenase Complex; an enzyme oxidizes Pyruvate to form Acetyl Co – A.
4. Cycle involves C6 to C4 in a series of steps:
Tricarboxylic Acid (C6 – 6 carbon compound) is formed by the combination of Acetyl Co – A (C2) and Oxaloacetate (C4).
Condensation process takes place between Oxaloacetate and Acetyl Co – A to form C6 compound.
Further the C6 compound catabolize to produce reduced equivalents (NADH, FADH2).
Oxaloacetate + Acetyl Co – A à Citrate (C6)
5. Eight enzymes are involved in 4 oxidations:
On each oxidation, number of carbo atoms present reduces gradually leaving out C4 compound. C4 the condense to form C6 and he cycle continues.
6. Two sources for Acetyl Co – A:
Acetyl Co – A is not only derived from Glycolysis. Fatty Acid oxidation also provides Acetyl Co – A as its direct end product which can be utilized by the Citric Acid Cycle.
7. A cycle with various intermediate forms:
Citric acid cycle forms various intermediates which provides substrates for different biosynthetic process in plants.
For Example: α – keto glutarate forms glutamate which is essential in Nucleic Acid formation
8. Citric Acid Cycle coupled with Electron Transport Chain:
The reduced equivalents from the cycle is directly transported to complexes present in the inner cell membrane of mitochondria to yield ATP.
Mechanism of Citric Acid Cycle
The Citric Acid Cycle has two steps:
1. Pyruvate Decarboxylation
2. Citric Acid Formation
3. TCA Cycle
Step: 1 Pyruvate Decarboxylation
Enzyme Complex involved – Pyruvate Dehydrogenase Complex (combination of enzymes, coenzymes and cofactors)
Pyruvate Dehydrogenase Complex (PDC) consists of 3 enzymes and 3 cofactors.
They are:
a. Pyruvate decarboxylase(E) + Thiamine pyrophosphate (CoF) – Complex 1
b. Dihydrolipopyl Transacetylase (E) + Lipoic Acid (CoF) – Complex 2
c. Dihydrolipopyl Dehydrogenase (E) + Flavin Adenine Dinucleotide (CoF) – Complex 3
The enzyme complex catalyses the oxidation reaction of Pyruvate to Acetyl Co – A inside the mitochondria with the release of CO2 and reduction of NAD → NADH2
Step: 2 Citric Acid Formation
Enzyme: Citrate Synthase This step is the 1st step of the Citric Acid cycle
I – Citric Acid, a six-carbon compound is formed by condensation and hydrolysis. Acetyl Co – A and Oxaloacetic Acid combines along CH3 and COO- group of acetyl Co – A and Oxaloacetic Acid respectively. On hydrolysis, Coenzyme A is released and Citric Acid is formed.
Step: 3 TCA Cycle
Citrate is the tricarboxylic compound from which the cycle proceeds.
II – Formation of Isocitrate
Enzyme: Aconitase
Enzyme aconitase mediates dehydration and rehydration of citrate molecule forming an isomer of citrate which is isocitrate.
III – α – Ketoglutarate Formation
Enzyme: Isocitrate Dehydrogenase
Dehydrogenase is associated with oxidation of the isocitrate to form α – ketoglutarate with a release of CO2 and formation of carboxyl group. This oxidation involves a reduction of NAD → NADH2. The six – carbon compound now becomes five carbon compound (C5).
IV – Succinyl Co – A synthesis
Enzyme: α – Ketoglutarate Dehydrogenase Complex
The enzyme functions similarly to that of Pyruvate Dehydrogenase accounts for the oxidation of α – Ketoglutarate to Succinyl Co – A. CO2 is released and NAD → NADH2. In this step 4 the five – carbon compound reduces to four – carbon compound (C4).
V – Succinate
Enzyme: Succinyl Co – A synthase
The mitochondrial matrix provides a phosphate group to be attached to the Co – A releasing Coenzyme from the compound. On hydration (i.e. addition of water) succinate is formed. ADP → ATP
VI – Fumarate
Enzyme: Succinate Dehydrogenase
The enzyme catalysis the formation of Fumarate by reducing FAD → FADH2. A double bond is formed between 2nd and 3rd carbon.
VII – Malate
Enzyme: Fumarase
Adding Fumarase and hydrating Fumarate results in the formation of Malate.
VIII – Oxaloacetate formation
Enzyme: Malate dehydrogenase
As dehydrogenase is coupled with formation of reduced equivalents, oxaloacetate is formed by reducing NAD → NADH2. This is the last and the fourth oxidation step in the TCA cycle.
Significance of Citric Acid Cycle
1. Citric Acid Cycle mainly provides ATP as energy. This is the important function of the citric acid cycle.
Acetyl Co – A + FAD + 3 NAD + ADP + H2O →
→ CO2 + ATP + 3 NADH + FADH + Co – A
One NADH = 3 ATP
One FADH2 = 2 ATP
ATP = 1
Total = 3(3) + 2 + 1 = 9+2+1 = 12
Citric Acid Cycle provides 12 ATP but glycolysis produces 2 pyruvate molecule the above calculation accounts only for one cycle of pyruvate.
Two cycle of Citric Acid then provides 2(12) = 24 ATP molecules
2. Citric Acid cycle is Amphibolic. Amphibolic means a process or a reaction which can be both Catabolic and Anabolic.
Catabolic nature is the breakdown of pyruvate for energy.
Anabolic nature is that the intermediates of Citric Acid Cycle can be used for the synthesis of amin acids, purine, porphyrin and pyrimidines
3. As Citric Acid cycle provides intermediates for synthesis of amino acid, purines etc., the cycle cannot run in a proper way.
To make the cycle to run in a proper way, many replenishing reactions are taking place to fill the missing intermediates. This process is called the anaplerosis.
4. Citric Acid cycle is the final common oxidative for energy production of carbohydrates, lipids and proteins.
Citric Acid Cycle: Animal vs Plant Cells
The main difference is the enzyme Succinyl Co – A in animals involves the formation “GTP” whereas plants involves in the formation of “ATP”.
Citric Acid Cycle Citations
- Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys . 2014 Apr;68(3):475-8.
- Impaired Citric Acid Cycle in Nondiabetic Chronic Kidney Disease. EBioMedicine . 2017 Dec;26:6-7.
- Citric acid cycle redux. Trends Biochem Sci . 1990 Nov;15(11):411-2.
- Citric acid cycle intermediates in cardioprotection. Circ Cardiovasc Genet . 2014 Oct;7(5):711-9.
- The key role of anaplerosis and cataplerosis for citric acid cycle function. J Biol Chem . 2002 Aug 23;277(34):30409-12.
- Regulation of leukocyte function by citric acid cycle intermediates. J Leukoc Biol . 2019 Jul;106(1):105-117.
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Growth Factors: Definition, Types, and Examples
Continue ReadingWhat are Growth Factors?
An irreversible, progressive change in a living being for a particular time according to the availabilities of the essential internal and external factors is growth.
In plant’s life term the growth is unlimited and continuous when optimal conditions are present.
Plant growth consists of development of the zygote to seed, on appropriate condition seed germinate to form root and shoot.
Proceeding from then seeds differentiates into different system of a plant bearing different functions.
Development and growth of a plant depend completely upon seed health and the external, internal factors aiding its growth.
Growth factors influence each stage of development by determining the availability of essential nutrients, climate etc., which are external and hormonal synthesis, physiological maintenance, availability of enzymes and substrate for metabolic process etc., are internal.
Growth efficiency is high when internal and external factors are available in essential level and are synchronous with each other enhancing the growth of an organism.
Type of Growth Factors
Growth factors are common for all plant species but the optimal requirement for each species differ making each species unique and distinct.
The common factors affecting the plant growth are divided into;
A. External Growth Factors
(i) Biotic Growth Factors: interaction of plants with other plants their response are all the biotic factors.
(ii) Abiotic Growth Factors: interaction of plants with external non – living factors playing crucial role in plant growth and development
B. Internal Growth Factors
(i) Genetic Growth Factors: the hereditary material made of nucleic acid which assigns different protein molecule for different functions are the genetic factors.
(ii) Physiological Growth Factors: Mainly includes the hormones and enzyme present within the body and the plant requirement.
A. External Growth Factors
External growth factors are components which influence the growth of plants from outside the plant body.
(i). Biotic Growth Factors: biotic factor which influences the plant growth from external sides are just interaction of plants to other plants and competition among themself for the survival of them. These are managed by certain behaviors namely;
Mutualism, Herbivory, Parasitism
a. Mutualism: the interaction between plants for their cooperative mechanism for survival and yielding benefit to each other.
Example: lichens
b. Herbivory: it is the nature of animals where it depends upon plants for survival. When excessive grazing tales place other organism inhabiting the same region has low survival rate.
c. Parasitism: interaction between two organisms where one benefits from the interaction others not and are deprived from nutrition making it hard to survival.
(ii). Abiotic Growth Factors: Factors which does not involve biologically but are essential for growth. Different factors that influence the plant growth are:
a. Climate: each plant has its suitable climate at which they grow well. The climatic factors include: Precipitation, Temperature, Humidity, Solar radiation, Wind velocity and Atmospheric gasses.
1. Precipitation: is of all forms such as Rain, Fog, Haze, Dew, Snow, etc., out of which rain fall is crucial in plant growth. Precipitation is completely dependent on the geography of a region. The slopes of Western Ghats receive high rainfall than the plains therefore considering the rainfall plantation crops are grown at the slopes of Western Ghats. Similarly drier regions support drought resistance plants such as sorghum, wheat, millet etc.,
2. Temperature: temperature is determined by the topography of the region. An ideal range for any plant is between 15° C and 38° C. But for the ideal plant growth each species is unique. The temperature also influences other body activity such as diffusion of the gasses, solubility of different materials, etc.,
3. Humidity: Humidity is the presence of water molecule n the form of water vapor. Higher the humidity lowers the rate of transpiration. Hence water accumulates causing decay of plants.
4. Solar Radiation – Light: Respiration is the process where the CO2 consumed to produce O2. This takes place by the process called photosynthesis. Incident of light is essential for synthesis of energy for plants survival. The radiation controlled the situation by determining the temperature of the region.
5. Atmospheric Gasses: in atmosphere the ratio of Carbon, Oxygen and Nitrogen must be balanced properly other wise imbalances can causes destruction to entire living system.
b. Edaphic Growth Factors: is noting but the specificities of soil for plant growth. The soil has several criteria namely: soil moisture, air, temperature, pH, mineral content, organic content and microorganism.
1. Moisture content makes the availability of nutrients to the plant easily attainable.
2. Soil Air provides aeration which removes any accumulation of undesirable gases, remove the dampness of the soil thereby providing an ideal environment which is free from infections.
3. Temperature, affects the physico chemical process taking place inside the soil and keeps the microbial activity under control and prevent microbial accumulation
4. Soil Organism, microbial growth is well associated with root nodules present in the plant where they fix atmospheric nitrogen to fulfil the nitrogen need for plants.
5. Other, factors associate well with the above factors provide an ideal internal environment for plant growth.
B. Internal Growth Factors
Factors which are present inside a plant body which are responsible for the growth of the plant.
The internal factors are a. Genetic factors and b. Physiological factors.
i. Genetic Growth Factor: the hereditary material becomes responsible for the blueprint of the organism, its productivity, survival capacity and others. In plant breeding technology new methods are employed to produce an ideal variety for better yield. Such efficiencies and capacities are present inside the cell and does not require other factors to promote it.
ii. Physiological Growth Factor: physiological factor is the hormonal and other factors which influence the plant growth. The growth of plants are dependent on the GROWTH REGULATORS, which are hormones which promotes or inhibits the growth.
These are done by 5 major hormones:
a. Auxins: enhance the cell elongation, enlargement, phototropism, geotropism, flower, root initiation, development of flower, root, fruit seeds etc.,
b. Gibberellins: affects the enlargement of organism, resisting the growth of plants as a whole.
c. Cytokinin: Cytokinin works along with auxin helps in cell enlargement
d. Ethylene gas: they diffuse easily throughout the body and ripens the fruit, seeds and are present in meristematic tissues,
e. Abscisic acid: intervene the growth promoting effect by reducing the growth promoting factors.
Growth Factors Citations
- The Pivotal Role of Ethylene in Plant Growth. Trends Plant Sci . 2018 Apr;23(4):311-323.
- Auxins as one of the factors of plant growth improvement by plant growth promoting rhizobacteria. Pol J Microbiol . 2014;63(3):261-6.
- Cytokinin signaling in plant development. Development . 2018 Feb 27;145(4):dev149344.
- Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci . 2020 May;63(5):635-674.
- Brassinosteroids: Multidimensional Regulators of Plant Growth, Development, and Stress Responses. Plant Cell . 2020 Feb;32(2):295-318.
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Electroporation – an Overview, An Efficient Electroporation...
Continue ReadingWhat is Electroporation?
The process of introducing nucleic acids (DNA or RNA) into eukaryotic cells by nonviral methods is defined as transfection.
Using various chemical, phospho-lipid or physical methods, this gene transfer technology is a powerful tool to study gene function and protein expression.
Transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., neagtively charged phosphate backbones of DNA and RNA) into cells with a negatively charged membrane.
Various chemicals such calcium phosphate and DEAE-dextran or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule and thus enable sucessful delivery inside the cells.
This makes it easier for the DNA or RNA:transfection reagent complex to cross the membrane, especially for phospho-lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer.
Physical methods like microinjection or electroporation simply punch through the membrane and introduce DNA directly into the cytoplasm. .
Electroporation Principle
In electroporation a high-intensity electrical field transiently permeabilizes the cell membrane, enabling uptake of exogeneous molecules from the surroundings.
This technique has been used to introduce nucleotides, DNA, RNA, proteins, carbohydrates, dyes and virus particles into prokaryotic and eukaryotic cells.
It provides a valuable and effective alternative to other physical and chemical methods for transfection.
Adopted from from BioRender
Requirements for Electroporation
Sterile:
1. Electroporation cuvettes
2. 6 well plates
3. Microtips
4. Growth medium
5. Cells (A 549 Cell line)
6. Trypsin EDTA
7. Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).
8. DNA
Non sterile:
9. 70% IPA
10. Cotton balls
11. Electroporator
Cell Preparation
1. One – two days prior to electroporation, transfer the cells into 25cm2 flasks with fresh growth medium so that they will be 50–70% confluent on the day of the experiment.
For most cell lines the cell density will be 2–10 x 10^6 cells/ flask; about 1–20 x 105 cells are needed per electroporation.
2. Trypsinize the cells as described in previous experiment and scrap the cells if required with scrapper and transfer the cells along with the medium in to 15 ml centrifuge tube, and centrifuge the cells at 1300 rpm for 7 min at 40 C.
3. Discard the supernatant and re-suspend the cells in the required quantity of PBS (Phosphate buffer saline)
4. Count the cells using Neubar’s chamber and calculate the volume of the cell suspension to be used in the experiment (2 x 10^6 in 200 μl)
Electroporation Procedure
1. Set the parameter required for electroporation likes voltage, time pulse, no. of pulse (V 75 to 200, Pulse length 15 to 30 mili seconds)
Note: This is variable from instrument to instrument and cell type
2. Add plasmid DNA to the cuvettes (5 to 15 μg)
3. Then add 200 μl of cell suspension (containing 2 x 10^6 cells) to the 0.2 cm cuvette and tap the side of the cuvette to mix.
4. Place the cuvette in the shockPod. Push down the lid to close. Pulse once
5. Immediately after the pulse, transfer the cells to a 6 well plate and add 1.8 ml fresh media
6. Rock the plates gently to assure even distribution of the cells over the surface of the plate. Incubate the plates at 37 oC in 5% CO2 in a humidified incubator.
7. Add suitable concentration of antibiotics after 24- 48 hrs.
8. Do not keep the cells out for longer time after electric shock.
9. Do not rock the plates forcefully.
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Apoptosis Assays: A Step by Step Complete...
Continue ReadingAbout Apoptosis
Annexin V is a phospholipid binding protein with high affinity for binding to phosphotidyl serine (PS), which is a phospholipid present exclusively in the inner leaflet of the cell membrane.
Annexin V does not bind to normal cells because of its inability to penetrate the lipid bilayer.
During apoptosis, due to changes in membrane permeability, PS is translocated to the outer surface of the membrane with which Annexin V binds with high affinity in the presence of Ca .
Detection of cell surface PS with Annexin V thus serves as a marker for apoptotic cells. Similarly, propidium iodide (PI), which specifically stains the dead cells, is used as a marker for necrosis.
Adopted from from BioRender
Apoptosis Principle Assay
In this assay, we are using fluorescently tagged Annexin V and PI as markers for apoptotic and necrotic cells respectively.
The number of annexin positive cells and PI positive cells are counted under the fluorescence microscope to estimate the extent of apoptosis and necrosis in response to specific drug treatment.
Materials Required for Apoptosis Assays
Non Sterile:
1. Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).
2. Annexin binding buffer (10mM HEPES (pH 7.5), 140mM NaCl, 2.5mM CaCl2)
3. Annexin V FITC (1mg/ml in PBS)
4. Propidium iodide (1mg/ml in PBS)
5. Fluorescence microscope with green and blue filters
Apoptosis Assay Procedure
1. Remove the medium and wash the cells with PBS thrice and once with annexin binding buffer
2. Add 100μl of annexin binding buffer and subsequently add annexin V-FITC (final concentration should be between 2 μg/ml)
3. Incubate the cells in dark at 37oC for 30 minutes
4. Add PI (Final concentration 1 μg/ml) and keep at RT for 10 minutes
5. Wash the cells twice with annexin binding buffer and observe under the fluorescence microscope
6. Capture atleast 5 fields for each treatment and don’t forget to take phase contrast images of all the fields.
Apoptosis Assay Calculation
Percentage of apoptotic cells = [no. of annexin positive cells/total no. of cells] x 100 Percentage of necrotic cells = [no. of PI positive cells/total no. of cells] x 100
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Growth Rates: Definition, Types, and Examples
Continue ReadingWhat is Growth Rate?
Growth is the fundamental characteristics of any organism which is irreversible, progressive and exponential.
Growth is a gradual phenomenon taking place at fixed interval of time and specific for a given species.
The growth mechanism of plant is significant than other organisms where the cells divide throughout their life having an unlimited growth.
Plants have a specialized region called meristem where cells get dedifferentiated to divide and increase the biomass of the plant.
The growth is a quantitative measure over time.
The growth in plants is measured by growth rate which is a measure of increase in growth per unit time.
Types of Growth Rate
Growth rate is of two types and are classified based on cell division
(i) Arithmetic Growth Rate
(ii) Geometric Growth Rate
(i) Arithmetic Growth Rate
In arithmetic growth, following a cell division only a single cell attains the capacity for further division, another cell gets differentiated and matured.
This follows for further division. The division is along one side increasing the length of the plant.
For example: Elongation of root. When plotting a graph for root elongation against time, a linear curve is obtained.
The linear plot indicates the growth rate was arithmetic in nature.
In Arithmetic growth is long one direction.
The Arithmetic Growth is expressed as:
Lt = L0 + rt
Lt : Length at time t
L0 : Initial length at time zero
r : Elongation per unit time
(ii) Geometric Growth Rate
Geometric growth type involves cell division were both daughter cell retains the capability for further division.
The growth is exponential and rapid for a particular period of time and when subjected to external and internal factors, their growth varies.
They represent the overall growth of a plant or a system at a particular period of time.
When plotted against time a sigmoid curve is obtained.
The sigmoid shape represents the rate od growth over a different period of time indicating different phases.
The four phases are: Lag phase, Log Phase, Diminishing Phase and Stationary Phase
1. Lag Phase: the initial growth period is referred as lag phase. In this phase each cell starts to divide continuously and make itself easily available to uptake of nutrients and increase cell mass. The phase involves gradual increase in cell growth.
2. Log Phase: the rapid cell growth period is the log phase. Under Favorable environmental condition the cell growth increases exponentially in large scale by the multiplication of cell division. Simultaneous nutrient input and maturation takes place in this stage. However, the cell division exceeds the maturation
3. Diminishing Phase: the cells start maturation providing a higher yield of cellular metabolites. The growth or new cell formation is confined to certain region of meristems which divides but to keep up with overall plant growth. Reduces the rate of formation of new cells
4. Stationary Phase: A final stage of plant growth where the meristematic regions constantly produce new cells and old cells are removed. This constant maintenance of cell cycle is the Stationary Phase.
This exponential growth can be represented as;
Wl = W0 ert
Wl = Final Size
W0 = Initial size
e = base of natural logarithm
r = growth rate
t = time
r = relative growth rate indicating the efficiency index.
Relative growth rate is the measure of given system per unit time.
This measure is compared with Absolute growth rate, a total measure of plant growth per unit time.
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Cell Lysis: an Overview, Definition, Types, and...
Continue ReadingWhat is Cell Lysis?
A cell is a biological living unit which is typically an enclosed space containing specialized components called organelles.
The inside of a cell is filled with a fluid called the cytoplasm and the entire cell shape is maintained because of the plasma/ cell membrane.
The cell membrane is semi-permeable and is made up of components that contributes to its structural integrity.
Bacteria also have a cell wall, which provides them with an additional layer of protection.
It is important for the cell to regulate its own functions and prevent any kind of compromise to its morphology.
Cell lysis refers to the breakage of the plasma membrane or the cell wall and leakage of the cellular contents, eventually resulting in cell death.
It is exhibited by both eukaryotic and prokaryotic cells.
Understanding cell lysis is necessary as it can not only help us comprehend the mechanism behind it, we can also exploit those mechanisms for experimental studies.
Lysis is brought about by specialised proteins which compromise the cell membrane, and in case of prokaryotes, the cell wall, or by external agents such as detergents or mechanical means.
Types of Cell Lysis
1. Cytolysis
Cytolysis occurs when a cell bursts due to an osmotic imbalance that has caused excess water to move into the cell.
2. Oncolysis
Oncolysis is the destruction of neoplastic cells or of a tumour.
3. Plasmolysis
Plasmolysis is the contraction of cells within plants due to the loss of water through osmosis.
4. Immunolysis
Erythrocytes’ hemoglobin release free radicals in response to pathogens when lysed by them.
Natural Cell Lysis
Cell lysis is exhibited by various types of cells and although the end result is cell death, this mechanism serves to benefit either the causative organism or the host organism.
Described below are some ways in which cell lysis takes place in nature.
a. Virus Mediated Cell Lysis
Bacteriophages are a type of virus which infect bacterial cells and use the latter for their replication and survival.
Bacterial cell lysis due to a viral attack is one of the ways in which the viral particles can be released from the host cell after multiplication.
Since the bacterial cell wall is composed of peptidoglycan (a polymer), specialized proteins called enzymes are released to disrupt the cell membrane and cell wall.
Holin, endolysin and spannin are three such enzymes involved in this mode of lysis.
Holins are the enzymes which control the timing of cell lysis.
They keep accumulating near the cell membrane and when the viral particles are ready to be released outside the cell, they cause the formation of holes in the bacterial cell wall.
Endolysins are the enzymes which can access the cell wall via these holes and actually attack the bonds between the building blocks of peptidoglycan of the cell wall, thereby degrading it.
Spannins disrupt the outermost membrane of the bacterial cell.
Single stranded DNA phages have certain genes that prevent the synthesis of peptidoglycan components and result in a weakened cell wall and causing lysis.
Significance: Viruses increase their infectivity by causing lysis of bacterial cells and releasing their progeny.
b. Cell Lysis in Cell Death Pathways
In mammalian cells, different intracellular pathways are activated when there is a bacterial or viral infection.
Such pathways lead to cell death as that can be beneficial to limit the infection since it would reduce the number of cells required by the foreign organism to invade.
Cell lysis, as described earlier, involves disruption of the cell membrane.
The cell membrane is made up of molecules called phospholipids, which are basically phosphate groups attached to a lipid molecule.
Infection in mammalian cells results in a process called inflammation, which is simply the activation of immune system.
Inflammation results in activation of specialised enzymes called caspases when a cell requires to go into ‘death’.
These caspases activate proteins which can bind to the phospholipids of the cell membrane, form pores and result in cell swelling and lysis.
Significance: Lysis of mammalian cells infected by bacteria/viruses causes reduced infection potential of the latter.
c. Immune Cell Mediated Cell Lysis
Immune cells such as T-cells have the property to recognize foreign bodies called antigens.
They release granules which contain proteins called perforins.
These attack the antigens, cause pore formation and result in bursting of the foreign cells.
Significance: Immune cells can directly kill foreign bodies via cell lysis.
Artificial Methods of Cell Lysis
Experimental research in biology requires studies on cellular components.
Hence, artificial methods of lysing cells have been developed.
Some of those techniques have been described below.
a. Osmotic Cell Lysis
Cells maintain their size due to their surroundings which contain fluids that prevent excess inflow (endosmosis) or outflow or water (exosmosis).
Transferring cells to solutions (example- sucrose) with a different concentration as compared to the cytoplasm can cause endosmosis, causing swelling and lysis.
b. Detergent Mediated Cell Lysis
Detergents are compounds which have both water loving and water hating components, and that makes them an ideal candidate to disrupt the cell membrane.
Example- SDS, Triton-X.
c. Physical Breakage
Beads and rotating blades can cause physical damage to the cell membrane and result in lysis.
Cell Lysis Disease: Hemolytic Anaemia
Red blood cells (RBCs) have a lifespan of 120 days.
In abnormal conditions such as pathogen attack or when the immune cells of the body mistakenly characterize RBCs as foreign cells, the cell membrane of RBCs get disrupted and they die before the end of their lifespan.
This drastically reduces RBC count in the body and results in anaemia.
Applications of Cell Lysis
Cell lysis is a widely used method for intracellular studies.
Proteins, DNA and RNA and extracted by a combination of lysis methods.
Industrially useful products generated in the intracellular space by micro-organisms are also obtained by lysing their cells.
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