Chapter 4- culturing and visualizing cells Flashcards
Robert Hooke
Used a microscope to look at a cork, coined the term cell since the rectangles he saw reminded him of monk cells.
Antoni van Leeuwenhoek
Observed microorganisms under a microscope- these became the first descriptions of live cells.
Schleiden and Schwann
Proposed that cells constitute the fundamental units of life in plants, animals, and single celled organisms.
Culturing definition
Isolated cells can be maintained in the laboratory under conditions that permit their survival and growth.
Advantages to culturing (3)
- Specific types of cells can be grown in culture, without other types of cells growing with them.
- Experimental conditions can be better controlled
- A single cell can be grown into a colony of many identical cells (clones). The clones are genetically identical cells.
Disadvantages to culturing
Cultured cells are not in their native setting- it’s not the same thing as the environment cells would have inside an animal.
Culture of animal cells requires (3)
Nutrient rich media, special solid surfaces, and for conditions to match the cell’s natural environment as closely as possible. This includes temperature, pH, and access to nutrients.
Culture medium
A nutrient rich liquid added to the dish/flask with the isolated cells.
How is temperature and humidity of the culture controlled?
The cultures are kept in incubators where temperature and humidity can be controlled.
How do researchers guard against the contamination of cell culture?
This includes adding antibiotics to the culture to reduce bacterial contamination and adding reagents to the culture medium.
Which nutrients does a culture medium need to supply? (4)
- 9 amino acids must be included in the media, as these amino acids can’t be synthesized by adult vertebrae cells.
- 3 other amino acids that are only made in specialized animal cells, so they are typically included in the media as well.
- Glutamine- serves as a nitrogen source
- Vitamins, salts, fatty acids, glucose, and serum (this is the fluid remaining after blood plasma clots).
Why is serum important for cultured cells?
Serum contains protein factors like insulin, transferrin (supplies iron), and growth factors. Certain cell types will require specialized growth factors that aren’t included in serum.
Why are cellular adhesion molecules important?
Most animal cells will only grow attached to a solid surface. CAMS bind to other cells or components of the extracellular matrix like collagen. The solid growth surface will usually be coated with extracellular matrix proteins so the cells will attach to them. These proteins can also come from the serum.
How long does it take for a cell colony to grow?
A single cell cultured on a glass or plastic dish will proliferate to form a mass (colony) in 4-14 days.
Primary cells
Cells isolated directly from tissues. Normal animal tissues or whole embryos are used to establish primary cell cultures.
How are cells prepared for a primary culture?
The interactions between cells and between the cell and the extracellular matrix must be broken.
To do this, tissue fragments are treated with a protease (like a collagen hydrolyzing enzyme) and a divalent cation chelator (which gets rid of free calcium in the medium). Once the cells are released, they can be put in dishes with the medium.
What is the importance of fibroblasts?
Fibroblasts are found in connective tissue. They produce extracellular matrix components, like collagen, that will bind to cells. Fibroblasts divide more rapidly than other cells and must be removed when other types of cells are being cultured.
Cell senescence
When cells are removed from an embryo or adult animal and cultured, the adherent cells will divide a finite number of times and then stop growing. Fetal fibroblasts will divide 50 times before stopping.
Cell strain
A lineage of cells originating from one initial primary culture. Cell strains can usually be frozen and resume growth after they thaw.
Under appropriate conditions, which cells are able to grow indefinitely?
Embryonic stem cells
How can cells be transformed so that they will grow indefinitely?
Cells that undergo an oncogenic transformation are able to grow indefinitely. This is observed in tumor cells, or in normal cells that mutate spontaneously. This is important because biologists want to be able to maintain cell cultures for more than 50 doublings.
Cell line
A culture of cells with an indefinite life span and is considered immortal. HeLa cells were the first cell line, and came from cervical cancer.
Aneuploid
Cells in immortal lines often have chromosomes with abnormal DNA sequences. These cells are said to be aneuploid.
A line with a single copy of most genes is useful for genetic analysis
Flow cytometer
Flows cells past a laser beam that measures the light they scatter and the fluorescence they emit. Therefore, it can quantify the cells in the mixture that are expressing the fluorescence protein.
What is the difference between a flow cytometer and FACS?
FACS can analyze the cells and separate them into another culture dish, a flow cytometer can only analyze the cells.
FACS
Fluorescence activated cell sorter- capable of both analyzing the cells and separating the fluorescent cells into another culture dish.
This procedure is frequently used to purify different types of white blood cells
Why is FACS used to purify WBCs?
Each white blood cell has distinctive proteins on its surface. The proteins will bind to their specific monoclonal antibodies. Biologists can add fluorescent dye linked to the antibody that goes with the specific cell surface protein. For example, only T cells have CD3 and Thy1 proteins, so they can be separated from a mixture based on these proteins.
Uses of flow cytometry (4)
- Measures a cell’s DNA content
- Determines the size and shape of a cell from the amount of scattered light
- Identifies specific cell types in a population
- Used to study signaling pathways in single cells.
How does flow cytometry measure a cell’s DNA content?
Measures a cell’s DNA content from the amount of fluorescence emitted from the DNA binding dye- these measurements are used to follow replication of DNA as the cell progresses through the cell cycle.
How does flow cytometry distinguish cell types in a population?
Identifies specific cell types in a population by using antibodies that are specific to a cell type. Fluorescent molecules are attached to the antibodies. Distinguishes cell types in blood samples
How is flow cytometry used to study signaling pathways in cells?
Stimulation of some signaling pathways causes the phosphorylation of specific proteins. Flow cytometry quantifies the phosphorylation levels of specific proteins in individual cells. Fluorescent reporter molecules are linked to phospho-specific antibodies. Flow cytometry will measure the phosphorylation of the proteins to assess the activation of signaling pathways.
How are epithelial cells grown in culture?
Many cell types function only when they are linked to other cells. Epithelial cells cover the internal and external surfaces of organs. They function by transporting molecules across the epithelial sheet. They are unable to do this when stored on plastic or glass. Therefore, there are containers with porous surfaces that act as a basal lamina. Epithelial cells can attach to it and form a two dimensional sheet.
Which cell line is used to study epithelial cells?
The Madin-Darby canine kidney (MDCK) cell line is used to study the formation/function of epithelial sheets.
How are cells able to grow three dimensionally?
Under appropriate conditions, MDCK cells can form a tubular structure that looks like the duct of a secretory gland or a tubular organ. The apical side lines the lumen, the basal side is in contact with the extracellular matrix.
How are adult stem cells different from embryonic stem cells?
Adult stem cells are necessary for tissue repair but are not pluripotent, like embryonic stem cells. Embryonic stem cells are able to give rise to any tissue. The division/differentiation of adult cells is controlled by many factors, like interactions with the cell matrix and other cells, response to soluble factors, and changes in gene expression.
How are adult stem cells used to make organoids?
Adult stem cells have been used to make intestinal organoids that had the crypt and villus structures of a normal small intestine. Brain organoids have also been developed, and have a similar structure to the fetal brain. Currently, organoids advance to the stage of development seen in a fetus but don’t continue to the organization seen in adult organs. These results show that cells have an intrinsic program to divide, differentiate, and organize themselves into complex structures.
Uses for organoids (4)
- Cells can be followed more easily in an organoid than in a living animal- we can examine early human brain development in more detail.
- The stem cells that are used to make organoids can be manipulated by introducing mutations and then seeing how they affect the development of an organoid.
- Might be able to test the effects of drugs on organoids, which is easier than it is for animals.
- Tumor cells from a patient can be used to make a tumor organoid in culture and use it to test the efficacy of drugs before they’re given to the patient.
Which problem of organ transplant can be resolved by organoids?
This solves the problem of immune rejection and can be used in the future to make synthetic organs.
Antibodies
Proteins secreted by white blood cells, and they have a high affinity for their antigen that they are secreted in response to.
Epitope
An epitope is a small region that contains a small amino acid sequence. Any antibody producing B lymphocyte is capable of making a single type of antibody that binds to a specific epitope on an antigen.
Polyclonal antibodies
Antigen exposure usually causes multiple different B lymphocyte clones that produce different antibodies. Most antigens contain multiple epitopes, and polyclonal antibodies are able to bind to different epitopes.
Monoclonal antibodies
Activated B lymphocytes form a clone of cells in the spleen or lymph nodes, and each cell produces identical antibodies. These antibodies will only bind to one epitope.
How are we able to produce monoclonal antibodies?
We need a line of immortal antibody producing cells. We can do this injecting an animal with an antigen and waiting for it to produce the B lymphocytes that will bind to that antigen. We extract these cells and mix them with multiple myeloma cells to form a hybridoma.
Hybridoma cells
These cells are screened with assays to determine which ones produce the monoclonal antibody of interest. Hybridoma cells are immortal, and each one produces the monoclonal antibody encoded by its B lymphocyte parent. We can grow the hybridoma cells indefinitely to mass produce the monoclonal antibody.
3 ways that hybridoma cells can be used
- They can be grown to mass produce the antibody of interest
- They can be injected into organisms to study cancer
- They can be frozen for later use
Which experiments can monoclonal antibodies be used for? (4)
- Affinity chromatography- to isolate and purify proteins.
- Immunofluorescence microscopy- to locate proteins in cells
- Immunoblotting to identify proteins in cell fractions
- They can bind to and inactive toxins in bacterial diseases
How are cell biological processes studied with cultured cells?
One way to understand a biological process is to interfere with part of the cell and assess the outcome- what will go wrong when a part of the cell is “broken”? Researchers can rely on naturally occurring genetic lesions like with genetic diseases, or they can manipulate cultured cells to interfere with the expression of specific components
How are drugs used in cell biological research?
We can analyze biological processes by treating cells with drugs that will bind to certain cell components and activate/inactivate them.
What is the resolution of a conventional light microscope?
2 micrometers
What are two major decisions to make when using a microscope?
- What microscope do I use?- all microscopes are designed for a different purpose
- How do I prepare the tissue/cells?
Resolution
The ability to distinguish between 2 closely positioned objects
How do we generate contrast in a microscope? (3)
- Dyes
- Manipulating light
- Computers
Categories of dyes (2)
- Calorimetric- absorb light and have color as a consequence
- Fluorescent dyes- absorb light- emit light at a different wavelength
Bright field microscopy/compound light microscopy
The oldest microscope technique, but the most popular. Uses calorimetric dyes. It has an objective lens and projection lens for viewing specimens, and the objective lens is used to collect light.
What type of experiment could bright field microscopy be used for?
Viewing live cells. However, these microscopes can’t see the fine details of cells, and the cell must be prepared so they can be visible. This is because live cells don’t absorb light.
Light microscopy protocol (5 steps)
The cells are fixed, dehydrated, embedded in paraffin, and sectioned using a microtome. They are then stained with a dye that allows the main structures to be visible.
What are fixatives used for?
Fixes the tissue as if it was in the lifelike state. Formaldehyde is a common fixative.
Formaldehyde
A fixative that cross links amino acids on adjacent molecules. It stabilizes protein-protein and protein-nucleic acid interactions and makes the molecules more stable for other procedures
How are cells/tissues dehydrated?
They are soaked in alcohol-water solutions. Ethanol is used to replace the water, and xylene is used to replace the ethanol- it is an organic compound that is compatible with the embedding medium.
Cryosectioning
This is an alternative to paraffin embedding. Samples can be quickly frozen in liquid nitrogen without dehydrating, and are then cut using a cryostat (similar to a microtome). It can be used to view the fine details of a cell under a microscope.
Cryosection applications
Allows for quick tissue biopsy in the operating room. Mohs surgery is used to treat skin cancer and takes a circular biopsy. Takes small amounts of tissue several times and views them using cryosections to determine if there are clear margins
Cryostat
Used for cryosectioning, basically a microtome but in a freezer
Phase contrast microscopy
An optical-microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. The darkness or brightness of a region of a specimen depends on the refractive index of that region, or how much the light bends when it passes through the specimen.
What is phase contrast microscopy used for?
It’s used for observing single cells or thin cell layers, but not thick tissues. It’s most useful for examining the location and movement of larger organelles in live cells
What causes fibrosis in the lungs of covid patients?
Due to the storm of chemicals from the virus- lymphocytes load the lungs. Fibrosis means that there is lots of collagen due to inflammation and the lungs aren’t able to exchange oxygen correctly.
DIC microscopy
The light is split into two components and recombined after it passes through the specimen to observe the interference pattern. It generates contrast based on the refractive index of the specimen and its medium. DIC images represent thin optical sections of a specimen, and a 3D image can be created when the sections are reconstructed.
What do phase microscopy and DIC microscopy have in common?
They are both used in live cell microscopy. This is where the same cell is viewed at regular intervals over time to generate a movie of cell movement.
How do the images from bright field microscopy, phase microscopy, and DIC microscopy compare?
In bright field microscopy, the cells are barely visible as no special methods have been used to generate contrast. In phase microscopy, there is a bright halo surrounding the image. In DIC microscopy, there appears to be a shadow to one side due to differences the refraction index.
What can DIC microscopy be used for?
It is most often used to view extremely small details and thick objects. It can define the outlines of large organelles as well. Also important for single cell electrophysiology, like with patch clamping
Hematoxylin and eosin stain
Hematoxylin binds to basic amino acids, and eosin binds to acidic molecules, like DNA and the side chains of aspartate and glutamate. Because they have different binding processes, these stains allow different types of cells to be distinguished visually.
Patch clamping
Patch clamp records the ion flux to a single ion channel in the cell membrane. Used for action potential readings in single cell electrophysiology. DIC microscopy is important for this.
Dark field microscopy
Describes an illumination technique used to
enhance the contrast in unstained samples. It works by illuminating the sample with
light that will not be collected by the objective lense and thus will not form part of the image. This produces the classic appearance of a dark, almost black background
with bright objects on it
Why use dark field microscopy instead of bright field microscopy?
Good candidates for darkfield observation often have refractive
indices very close in value to that of their surroundings and are difficult to image
with conventional bright field techniques
Applications of dark field microscopy
Used for live, unstained specimens. Used in transmission electron microscopes, imaging of atoms and other small particles, as in microbiology.
Polarizing light microscopy
Polarizing light to analyze “highly ordered parallel structures”. Ex- collagen, microtubules, and microfilaments. A light source going through a polarizer creates polarized light (sort of like sunglasses work). Light goes through an analyzer and we can see the highly ordered structures in cells. This method is qualitative.
What is polarizing light microscopy used for?
Can be diagnostic for looking at fibrosis- the collection of collagen fibers present in diseases. These fibers are highly ordered.
Qualitative
We get an image
Two major limitations of conventional fluorescence microscopy
- The observer sees a blurred image due to the superposition of fluorescent images from molecules at different depths of the cell
- For thick specimens, images must be taken at different levels of the specimen and be reconstructed.
Deconvolution microscopy
Uses a computer to remove fluorescence from the out of focus parts of the sample and increase practical resolution.
Confocal microscopy
A microscope that has a microlens array disc incorporated into it and is designed to
visualize dynamic changes in cells revealed by fluorescent dyes with a minimal level of
photobleaching. They collect a series of vertical images and use them to create a 3D representation of the sample. Includes spinning disk and laser scanning confocal microscopy.
Point scanning confocal microscopy
Uses a point laser light source at the excitation wavelength to scan the focal plane, collect the fluorescence in a photomultiplier tube, and create a high resolution 2D or 3D image.
Limitations of point scanning confocal microscopy (2)
- It takes a lot of time to scan each focal plane, so this isn’t a great technique for imaging dynamic cell processes
- Each spot is illuminated with intense laser light, which can bleach the fluorochrome and damage the cells by phototoxicity
Spinning disk confocal microscopy
Circumvents the limitations of point scanning microscopy. It spreads out the light from the laser and illuminates a small part of the disk spinning at high speed. It uses moving pinholes in the disk that are used to scan the focal plan. This method is best for imaging the processes of live cells and is much quicker than point scanning.
Limitation of spinning disk confocal microscopy
Pinhole size is fixed and has to be matched to the magnification of the objective lens, so it’s less useful for lower magnification imaging.
Advantages of confocal microscopy (4)
- Increase in practical limit of resolution
- Decrease in stray image
- Stereo imaging and “Z stack”- double and triple labeling cells
- Can see a single cell
Caliber ID Vivascope
A handheld confocal microscope used to detect skin cancer- noninvasive analysis of skin irregularities/skin cancer. Advantage- optical section for cellular imaging. . Point of care diagnosis- your dermatologist can evaluate it in that way.
Fluorochrome
Fluorescent dye that is colorimetric- eg hematoxylin and eosin. A chemical is fluorescent if it absorbs light at an excitation wavelength and emits light at a longer wavelength.
Vital fluorescent dyes
monitor changes in cell physiology in living cells during real time. Mitochondrial function can be analyzed using fluorochromes. If the mitochondria are green, it indicates a low proton motive force. This means the mitochondria aren’t very active and aren’t making a lot of ATP. These dyes are also used for live-dead assays
Live-dead assay
Calcein-AM. During cell death, the cell membrane breaks and calcein comes out. Indicates membrane integrity. Propidium Iodine- red dye- not membrane soluble. It will be able to get into the membrane if the membrane is compromised and it will stain the nucleus red. This technique can be used to measure necrosis.
Cytofluor
First plate reading spectrofluorometer
Fluorescence microscopy
These microscopes allow the excitation light to pass through the objective lens into the sample and allow the user to observe the emitted fluorescent light coming back through the objective lens from the sample.
Fluorescence recovery after photobleaching (FRAP)
Uses high intensity light to permanently bleach the fluorochrome. If the bleached molecules are in equilibrium with unbleached fluorescent molecules elsewhere in the cell, the molecules with diffuse and the bleached molecules will be replaced with fluorescent ones. The rate of fluorescence recovery is used to measure the dynamics of molecules.
What is FRAP used for?
Can be used to monitor the lateral diffusion of a protein of interest
Total internal reflection fluorescence (TIRF) microscopy
Only a portion of the specimen immediately adjacent to the coverslip is illuminated. If a specimen has a complex mixture of fluorescent structures, TIRF can be used to only show structures near the coverslip.
What is TIRF used for?
Can identify structures on the bottom of cells grown on a coverslip. Measures the kinetics of assembly of structures like actin filaments
Intracellular injection of living cells using fluorochromes
Used to trace synaptic connections of
neurons (Lucifer yellow dye). Can identify gap junctions, a gap junction is present if the dye moves to an adjacent cell
Fluorescence immunocytochemistry
Using antibodies and fluorochromes to detect the presence of proteins in cells. Monoclonal antibodies are produced and tagged with a fluorochrome, then the antibody will bind to the antigen and light up when exposed to the excitation wavelength.
Indirect fluorescence immunocytochemistry
Primary antibody is bound to antigen which is then amplified by
use of a fluorochrome tagged secondary antibody. This allows two proteins to be visualized simultaneously.
ELISA
Enzyme Linked Immunosorbent Assay – used to measure the concentration of
antibodies or antigens in a solution, uses both primary and secondary antibodies -
not a microscope technique. The antibodies will bind to the antigens of interest and the unbound antigens will be removed.
Apoptosis
“suicide”- programmed cell death. This is how chemo and radiation work.
Necrosis
pathological cell death- “murder”, like how ricin poisons ribosomes
How is apoptosis measured?
Annexin V is a probe that is used to determine if a cell has committed to apoptosis. Annexin V stains the plasma membrane when phosphatidylserine is exposed to the outside surface of the cell – labels cells that are proceeding through apoptosis
DNA gel for necrosis/apoptosis
Distinct bands if apoptosis,
smear if necrosis
Green fluorescent protein (GFP)
GFP is found naturally in jellyfish. It is used to tag a protein of interest so that protein’s location can be visualized.
GFP applications
We can view the distribution of the protein in a live cell over time and assess its dynamics or we can view its localization after a treatment, like with a specific drug. In human-pig chimera embryos, GFP labeled human cells can be tracked.
YFP
Yellow Fluorescent Protein – genetic mutant of GFP. Acceptor for genetically-encoded FRET sensors. Reduced chloride sensitivity, faster maturation, increased brightness are
improvements
Forster Resonance Energy Transfer (FRET)
Fluorescence microscopy technique, uses a pair of fluorescent proteins in which the emission wavelength of the first is close to the excitation wavelength of the second. One fluorescent protein will fluoresce, and the second protein will be excited by that wavelength if it is close by.
FRET applications
Measures the distance between proteins or signaling between proteins
FRET biosensor
A single polypeptide containing both CFP and YFP is used, but the proteins are separated by a region that undergoes a conformational change when it senses a biochemical signal. When the signal is detected, the conformational change occurs and brings the proteins close enough together that FRET will occur.
FRET biosensor applications
Used to sense local biochemical environments in live cells. A biosensor has been developed for protein kinase A activity.
Autoradiography
Use of a radioisotope to monitor a change in cell physiology. Radioactivity will develop film thus seeing which cell is decaying faster or at all. DNA synthesis, protein synthesis are uses.
2 examples of autoradiography
- 3H-thymidine- DNA base with 3H= radioactive. Add to living cells, 3H- thymidine goes to the nucleus.
- S-methionine or 3H-leucine (both amino acids). Newly synthesized proteins- used to track protein synthesis.
Fluorescence In Situ Hybridization (FISH)
Uses fluorescent single stranded nucleic acid probes that bind only to
those parts of the gene or RNA that are complementary.
FISH applications (3)
- Telomere labeling
- Binding to a target mRNA in the cytoplasm
- Can be used to test for the HPV gene
Intracellular injection techniques (4)
- Single cell microinjection- used in neurobiology
- Electroporation- applies voltage to cells to make their membranes more permeable
- Liposomes and nanoparticles- liposomes are phospholipid membrane systems, critical to Pfizer/moderna mRNA delivery
- Viruses
Transmission electron microscopy (TEM)
Beam of electrons transmitted through sample to form image on photographic plate/screen- can view internal structures. Gold sections and copper rids are most associated with this technique (copper used in the making of the stage for the sample, gold sections to bind to sample in desired
regions
Limitations of TEM
Samples must be cut into very thin sections, can’t be used to image living material.
Plastic thin sectioning
Used for electron microscopy. Fix larger specimen into thin size needed to be viewed. There is a process of chemically fixing the sample, dehydrating it, and impregnating it with a liquid plastic that hardens before sections can be cut. Lead is used to stain membranes, and uranium is used to stain everything else.
Freeze fracture stain
Freeze membrane and split it along middle of the phospholipid bilayer. Allows one to see cell membrane structure. Combinations used includes platinum replica, carbon coating
Ultrastructural autoradiography
Uses radioisotopes, reveals the 3D nature of the internal structure of a cell
Ultrastructural immunocytochemistry
Gold particles attached to antibodies that attach to cells. This is done so particles can label what will be seen by electron microscope. The tag most closely linked to ultrastructural immunocytochemistry is gold (attached to
antibody) Can be used for LDL internalization
Scanning electron microscopy
Allows investigators to view the surfaces of unsectioned metal coated specimens. An electron beam scans the sample. Surface and shape viewed, used for analysis of conductive materials only
High voltage electron microscopy
Whole cell imaging, high accelerating voltage. Heat is a problem
Affinity chromatography
This is a column technique that relies on the fact that certain proteins will bind to other proteins in a specific manner. A ligand cross links to the beads, then membrane proteins are added to bind or not to bind. The bound proteins are removed using a high salt concentration. To remove the salt, go through a desalting column.
Applications of affinity chromatography
Antibody affinity chromatography, where antibodies will bind/not bind to a protein of interest. Can also use insulin as a ligand and see whether the insulin receptors (membrane proteins) will bind.
Gel electrophoresis
Separates proteins using a polyacrylamide gel. The gel is cross linked, like mesh. The proteins are drawn through the gel because they are negatively charged.
Native gel electrophoresis
Separates proteins in their native states, no protein preparation is necessary. Proteins are separated based on charge and size- proteins with a low molecular weight will move through the gel faster than proteins of a high molecular weight. The bands that are generated can be diffuse, so this technique is lower resolution.
Drawback of gel electrophoresis
Drawback- proteins can change their conformations or other properties when exposed to an electric field.
SDS gel electrophoresis
Similar to native gel electrophoresis, but the proteins are pretreated so they are denatured (not in the native state). Proteins with mutiple components can be separated into monomeric components. This technique gets better protein separation and is higher resolution, but the native protein activity can’t be preserved.
In SDS gel electrophoresis, which compounds are used to denature the proteins? (3)
- Beta-mercaptoethanol hydrolyzes disulfide bonds that bond parts of the protein together
- Urea disrupts the bonds between nucleotides (hydrogen bonds)
- Sodium dodecyl sulfate (SDS) is a detergent that coats the protein and makes them more negative.
Dimer
Two different proteins tied together
Isoelectric focusing
Gel electrophoresis technique, proteins migrate through a pH gradient to the pH where they reach their isoelectric point- they will then stop moving. Very powerful technique, blood serum for example can be separated into 40 bands.
Isoelectric point
The pH where a protein is considered electrically neutral. With isoelectric focusing, proteins are separated by isoelectric point rather than weight.
2D gel electrophoresis
Has the greatest resolution of all gel electrophoresis techniques. It uses isoelectric focusing, and these bands are used as samples for the next technique- SDS gel electrophoresis. Can be used for identifying a protein spot, sequencing it, and then identifying the gene that it encodes for
SELDI-TOF/MALDI-TOF
Surface-Enhancer Laser Desorption/Matrix Assisted Laser Desorption Ionization-Time of Flight - Used
for mass spectrometry of proteins, time of flight is based on the principle that smaller proteins “fly” faster than big proteins. Key parts of the system include a laser, chips, and TOF detector.
SELDI-TOF/MALDI-TOF advantages (2)
- Very small sample amount necessary- one microliter
- Resolution is one amino acid- no other system has this type of resolution
Western blots
Both a quantitative and a qualitative protein analysis system- determines the molecular weight of a protein of interest and its relative abundance. Uses gel electrophoresis (typically SDS) to generate bands and put them on transfer paper. You use a monoclonal antibody that will bind to an epitope within a protein of interest. The proteins are identified based on their ability to bind to antibodies.
Applications of SELDI-TOF/MALDI-TOF
Critical to the future of personalized medicine and protein profiling, can be used for Alzheimer’s detection. Alzheimer’s signature protein is amyloid beta 1-42, people without Alzheimer’s have amyloid beta 1-40. Calculating the ratio of the two can be diagnostic of predisposition or disease progression.
Western blots and photoreactive amino acids
Technique used to determine protein-protein interaction. Uses photoreactive amino acids as a probe (like leucine and methionine). Uses UV light and photoreactive amino acids to covalently link a ligand and a receptor. Then, one can do a western blot and note a band shift to a higher molecular weight indicating that the two are now covalently bonded.
Western blots and photoreactive amino acids applications
Can be used to determine whether protein X binds to an unknown protein Y. UV light activates the photoreactive amino acids to form a covalent bond between a ligand and receptor and “capture” protein Y. Western blots will then be used to
Protein A immunoprecipitation
Technique of precipitating a protein antigen out of solution using a monoclonal antibody that specifically binds to that particular protein. Isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Agarose has very high potential binding capacity
Atomic force microscopy
High- resolution type of scanning probe microscopy, with demonstrated resolution
more than 1000 times better than the optical diffraction limit, doesn’t use lenses. Can view DNA, cell pores, and membrane structure. Produces a 3D image.
Laser-capture microdissection microscopy
Laser to separate cell of interest so it can be analyzed closely. The LCM technique allows differentiation between normal and morphologically abnormal cells as distinct cell populations from the heterogenous mixture, and it is possible to investigate subcellular profiles with great accuracy.
Scanning Tunneling microscopy
Works by scanning a very sharp metal wire tip over a surface. By bringing the tip very close to the surface, and by applying an electrical voltage to the tip or sample, we can image the surface at an extremely small scale – down to resolving individual atoms. First to image native DNA.
Two photon microscopy
A fluorescence imaging technique that allows the visualization of living tissue at depths unachievable with other types of microscopy. It relies upon the principle of two-photon absorption- the concept that two photons of identical or different frequencies can excite a molecule from one energy state (usually the ground state) to a higher energy state in a single quantum event. This requires a focused laser that is able to produce very fast pulses of light
Advantages of two photon microscopy (3)
- Deeper tissue penetration
- Efficient light detection
- Reduced phototoxicity
Super Resolution Microscopy (3 types)
Much higher resolution than even a confocal microscope, not diffraction limited. Takes time to use, so unable to image living cells.
1. Photo-activated localization microscopy (PALM)
2. Structured illumination microscopy (SIM)
3. Stimulated Emission depletion microscopy (STED)
Photo-activated localization microscopy (PALM)
Collect thousands of images in each of which only a few GFP molecules are excited
Structured illumination microscopy (SIM)
Collect images with illumination
pattern in different orientations. Generate interference patterns than can be computationally reconstructed to create
an image
Stimulated Emission depletion microscopy (STED)
Excitation laser point
surrounded by donut shaped “depletion beam,” making area excited much smaller
Velocity sedimentation
Separate cells using gravity and based on their size. When layered over dilute solution, large cells sink to bottom first. Doesn’t use fluorochromes.
Dynabeads
Magnetic beads used to separate cells and isolate them, beads have an iron core or antibody receptors. Dynabeads and similar devices of this type that use selective surfaces are often used for cell and organelle separation, could also be used for cell culture growth substrates.
Veridex CellSearch System
Machine that uses magnetism or antibodies to sort out circulating tumor cells. Can use these to quantify an individual’s tumor burden
Differential centrifugation
Separation of cell components using gravity, the largest components are separated first. 1st time separates whole cell and nuclei, 2nd time separates lysosomes, 3rd time microsomes are finally separated out
Equilibrium Density (Rate Zonal) centrifugation
Method of cell fractionation in which you purify the organelle of interest that contains the protein of interest. All the proteins start from the thin layer of the sample that was placed at the top of the tube and separate into bands (actually, disks) of proteins of different masses as they travel at different rates through the gradient. In this separation technique, called rate-zonal centrifugation, samples are centrifuged just long enough to separate the molecules of interest into discrete bands, also called zones
Guava
Counts cells using fluorescence, doesn’t separate cells
DEAE and CM Ion exchange chromatography
Separates proteins based on charge. DEAE ion exchange chromatography is more commonly used than CM ion exchange chromatography because most proteins have an overall negative charge
(CM should be used for proteins with a positive charge). Negatively charged beads will allow negatively charged proteins to move through and positively charged proteins will bind.
Densitometry
Image analysis of a dried gel, scans each lane and converts it to relative densities. These measurements are converted into a graph to give relative abundance of a protein. Qualitative method.
In vitro, in vivo, in situ
In vitro= under glass (in culture)
In vivo= in the living animal
In situ= in place
CHO cells
Chinese hamster ovary cells are preferred for production of monoclonal antibodies, along with HeLa cells
Edible mRNA vaccines
Purpose of this project is to show that DNA containing mRNA vaccines can be successfully delivered to plant cells, and that plants can produce enough mRNA to rival a traditional vaccine. Genes are inserted into spinach and lettuce chloroplasts, not the nucleus.
Importance of the Golgi stain
Used a silver stain technique, discovered that brain tissue was actually made of separate cells. Randomly stained select cells.
George Gey
Grew HeLa cells in culture medium- did this without Henrietta Lack’s consent, one of the things that led to GINA.
Hayflick hypothesis
Typical human cell strains will divide for only 40 to 60 times and then enter senescence
Limitations of cell culture (6)
- Artificial environment doesn’t represent what would be found in vivo
- Cells grown in culture can change their genotype over time- immortal cell lines can undergo genetic drift
- Cells don’t always exhibit the proper phenotype in vitro unless plated with a substrate that supports their unique phenotype
- Not all cells divide in culture such as adult hepatocytes
- Many human embryonic stem cells require highly specialized media, making controlling the environment more complicated
- Function can be lost over time, without a loss in viability of cells- human hepatocytes
Microporous membranes
MDCK (Madin Darby Canine Kidney) cells must be grown on a microporous membrane to exhibit their appropriate phenotype. This is an example of a cell culture limitation
OB/OB mice
One mouse is obese, the other isn’t- the mouse that isn’t fat has a leptin patch to control appetite. These mice are transgenic
Microspectrofluorometry/Cytofluor
Qualitative (picture of image) use fluo 3-AM- intracellular calcium. Measures fluorescence based on increase in calcium concentration, and shows areas of high and low concentration. Cytofluor is quantitative.
Electron tomography
Reveals the 3D nature of the internal structure of a cell, used to get 3D structure of subcellular macromolecular objects
Pulse-chase
Method to examine cellular process over time and track protein movement in the cell. Pulse is radioactive label that is used to track process, chase is non- radioactive wash that flushes out the pulse
Radioactive tagging
Takes 28-30 days, form of isotopic labeling. Chemical compounds in which one or more atoms have been replaced by a radioisotope so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by tracing the path that the radioisotope follows
from reactants to products. Allows scientists to observe its movements within a living cell
EGTA
Protease inhibitor. Proteases are enzymes that break down protein.
Trituration
A process used to purify crude chemical compounds containing solid impurities. A solvent is chosen in which the desired product is insoluble and the undesired by-products are very soluble (or vice versa). The crude material is washed with the solvent and filtered away, leaving the purified product in solid form and any impurities in solution.
Gel filtration
Column technique, separates proteins based on size. Use beads to separate proteins (beads have holes in them). Larger proteins won’t fit through holes and pass through column, small proteins go through all of the holes and pass through column much slower
Dialysis
Dialysis is a separation technique that facilitates the removal of small, unwanted compounds from macromolecules in solution by selective and passive diffusion through a semi-permeable membrane. Sample molecules that are larger than the membrane-pores are retained on the sample side of the membrane, but small molecules and buffer salts pass freely through the membrane, reducing the concentration of those molecules in the sample.
Fetal bovine serum
Provides growth factors in a medium for when cells are in culture, Problems- expensive,
have contaminants- viruses, prions, etc
Defined medium
No serum required, growth factors are added
Biological scaffolds
Required for tissue engineering. There are two types: biologically degradable- made to disappear over time, and non biodegradable
Tissue engineering
The ability to use cell cultures to generate tissue engineered constructs (lab grown tissues). One requirement is typically some type of scaffolding to build the 3D tissue. The best cells to use are stem cells
Bioprinting/3D printing
Bioprinting scaffolds (biodegradable) part of tissue engineering , (stem cells). Can print tissue by swapping ink jet printer for suspension of cells. Rapid prototype - Group of techniques used to quickly fabricate a scale model of a
physical; part or assemble using 3D computer aided design
Decellularization
Decellularization is the process used in biomedical engineering to isolate the extracellular matrix (ECM) of a tissue from its inhabiting cells, leaving an ECM scaffold of the original tissue, which can be used in artificial organ and tissue regeneration
Examples of tissue engineering constructs (6)
- “Nose organoid”- application is for Covid, used to figure out how the virus gets into the nasal passages
- “Lung organoid” used to study covid infections in the lungs- want to see what can be used to block the virus binding to the ace2 receptor
- Human bladder- children with spina bifida often have bladder problems, a human bladder would allow for bladder implants
- ELAD= engineered liver assist device. Located outside the body, used to give the liver a break and let it heal
- Carticel- “Replacement” cartilage
- Heart patches- tested as a cardiac bandaid
Human skin tissue engineering construct
Made from NHEK- normal human epidermal keratin cells. Source- foreskin- male cells
Disadvantage of cell lines
Don’t behave like normal cells do. Ex- cancer cells have p-glycoproteins unlike normal cells
DNA microarrays
Uses glass slide, fluorochromes are used in association with probes. Requires hybridization, and 6000 to 8600 different nucleotide sequences are often critical to this technique. Responsible for cluster analysis to screen for genetic mutations
Stem cell culture has traditionally required what type of substrate?
Feeder cell layers
Why are monoclonal antibodies preferred over polyclonal antibodies?
They can be selected for high specificity and affinity and the cells generating the
monoclonal antibody can be stored frozen in liquid nitrogen.
IPTG, an agent that behaves like lactose, is important in cell expression systems. Why is this the case?
It can turn on or off the lac operon that can be genetically manipulated and used for
prokaryote gene expression.
A clinical example of the use of Southern Blots and VNTRs was discussed in class in
reference to bone marrow transplant. What was the overall importance of this
experiment?
It showed that no residual cancer cells remained in the patient after donor cell
transplantation
VNTRs
VNTR are structural regions of the DNA where a short sequence of nucleotides (longer than 3) is repeated a variable number of times in tandem. They are thought to be due to DNA slippage errors during DNA replication.
Southern blots
A laboratory method used to study DNA. Purified DNA from a biological sample (such as blood or tissue) is digested with a restriction enzyme(s), and the resulting DNA fragments are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.
Northern blots
A laboratory analysis method used to study RNA. Specifically, purified RNA fragments from a biological sample (such as blood or tissue) are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The RNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent or chemical tag. The tag allows any RNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Northern blot.
Why is cytochrome p450 enzyme activity so important?
This was referenced twice in lecture- in one case it was with human primary hepatocytes growing in culture and the other case was in reference to C3A cells in ELADs. Its activity is a measure of the cells’ detoxification function
Protein or gel shift assays are used for what purpose?
Determine which protein within a mixed group of proteins binds to a select nucleotide
sequence such as a gene
Complementation analysis is a technique commonly considered to belong to which group of techniques?
Molecular biology/gene identification
Lucifer yellow is used for
It’s a fluorescent dye used in conjunction with neural tissue or ganglions
(group of neurons). It can perfuse through the cell and, as such, outlines the entire length of the cell into which it is injected
What is the size of a typical mammalian cell?
10 micrometers
Transmission electron microscopy techniques (4)
- Plastic thin sectioning
- Freeze-fracture stain
- Ultra structural immunocytochemistry
- Ultrastructural autoradiography
Telomeres
The tips of chromosomes. They are referred to as the “mitotic clock” and are part of the reason why cell lines can divide an infinite number of times. In immortal cells like cancer cells, telomeres don’t shorten.
How are Guava and Cytofluor different?
The Guava can count cells one by one and present them as a dot plot
JC-1
Fluorescent dye that is used in apoptosis studies to monitor mitochondrial health.
The resolution of a transmission electron microscope is primarily governed by
The speed of the electrons illuminating the specimen
Which microscope is not diffraction limited?
Super resolution (resolved) fluorescence microscopy
