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BIOL 4610 > Module 1 Part 1 > Flashcards

Module 1 Part 1 Flashcards

(60 cards)

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1
Q

What are primary cells?

A

cells isolated directly from tissues; most closely represent the tissue of origin

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2
Q

How is a primary cell culture formed?

A
  1. Treat tissue with disrupting agents (proteases and EDTA)

2. Place the released cells in a nutrient rich medium in a dish where they can adhere to the surface and one another

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3
Q

What do proteases do?

A

Proteases such as trypsin and collagenase are proteolytic enzymes that cleave protein-protein interactions.

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4
Q

What does EDTA do?

A

EDTA is a calcium chelator that bonds to calcium ions in order to disrupt CAMS. By disrupting the CAMS, the cells are released from the tissue and the solid growth surface.

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5
Q

What are CAMS?

A

CAMS (cell adhesion molecules) are proteins in the plasma membrane of cells that bind to similar proteins on other cells and proteins in the extracellular matrix. They require calcium to be functional.

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6
Q

What is senescence?

A

the phenomenon in which cells derived from a tissue will eventually stop growing (have a finite number of divisions)

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7
Q

What is a cell strain?

A

a population of cultured cells that has a finite life span and eventually dies, commonly after 25-50 generations

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8
Q

What is a cell line?

A

a population of cultured cells that has undergone a genetic change allowing the cells to grow indefinitely
ex: HeLa

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9
Q

What conditions are required for culturing cells in the lab?

A

trying to simulate the conditions within an intact organism as best as possible including:

  • pH
  • ionic strength
  • rich medium with 9 essential amino acids, vitamins, peptide and protein growth factors
  • antibiotic
  • antimycotic
  • negatively charged solid surface (glass or plastic) to mimic extracellular interactions in animal cells
  • CAMs
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10
Q

What is a “passage of cells”?

A

subculture or splitting of confluent cells into a new culture

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11
Q

What is a disadvantage of using primary cells?

A

Primary cells have a finite number of doublings (50 to 100) until they die.

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12
Q

What is a disadvantage of a cell line?

A

In a cell line, the cells are immortal, but they may not accurately represent the original cells in the tissue because they are genetically altered.

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13
Q

How are embryonic stem cells grown?

A
  1. Cleavage-stage embryo is developed by in-vitro fertilization into a cultured blastocyst
  2. Inner cell mass (ICM) is separated from surrounding tissue
  3. ICM is placed in contact with fibroblast feeder cells
  4. Dissociated cells are re-plated
  5. ES cell cultures are established
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14
Q

Why is embryonic stem cell research controversial?

A

The blastocyst is destroyed when the inner cell mass is isolated. Therefore, in order to study embryonic stem cells, you have to destroy an embryo.

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15
Q

How can embryonic stem cells be grown without feeder cells?

A

The culture would require specific cytokines such as leukemia inhibitory factor, Wnt, and bone morphogenic protein 4. This ensures that the physiological characteristics measured from the embryonic stem cells belong to them alone and not the feeder cells.

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16
Q

What physical property can cells from a heterogeneous population be separated by?

A

density (using centrifugation)

ex: red and white blood cells

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17
Q

What physiological property can cells from a heterogeneous population be separated by?

A

affinity for glass

ex: microphages

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18
Q

How does flow cytometry work?

A

In flow cytometry, you can stain T cells with fluorescent antibodies (red fluorescence conjugated anti-CD3 antibody and green fluorescence conjugated anti-Thy1.2 antibody) that only T-cells will bind. The cells are then separated using a fluorescence-activated cell sorter (FACS). The T cells separated should have a high level of both green and red fluorescence.

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19
Q

What are the surfaces of epithelial tissue?

A

apical surface, lateral surface, and basal surface

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20
Q

How can epithelial cells be grown to mimic epithelial tissues?

A

Epithelial cells can be grown on special containers designed with a porous surface that act as a basal lamina. The epithelial cells attach to the basal lamina and form a uniform 2D sheet. The apical surface faces the lumen and the basal surface faces the basal medium. A Madin-Darby canine kidney (MDCK) cell line is often used.

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21
Q

How do hybridomas produce abundance monoclonal antibodies?

A
  1. Mouse is injected with antigen X
  2. Mutant myeloma cells are fused with mouse spleen cells (some of which have made the antibody to antigen X
  3. Fused cells are transferred to selective HAT medium
  4. Unfused cells die and fused cells grow
  5. Single cells are cultured in separate wells and tested for the antibody to antigen X
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22
Q

What is in the HAT selection medium?

A

Hypoxanthine-Aminopterin-Thymidine

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23
Q

What does aminopterin do?

A

Aminopterin blocks the enzyme dihydrofolate reductase. This blocks the De Novo pathway of making DNA.

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24
Q

What is the De Novo pathway of making DNA?

A

Sugars + amino acids -> Nucleotides -> DNA

blocked by aminopterin

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25
What is the Salvage pathway of making DNA?
Nucleosides + TK + HGPRT -> Nucleotides + DNA
26
Which cells with survive and which cells will die during the HAT selection of hybridomas?
Unfused myeloma cells die because they do not have Tk or HGPRT and can not go through the Salvage pathway to make DNA. Fused B- and myeloma cells survive and are immortal. These produce monoclonal antibodies and can be purified. Unfused B-cells die because they are mortal. They can still produce DNA using the Salvage pathway, but they have a limited number of replication cycles.
27
What is an epitope?
The part of an antigen molecule that binds to an antigen-specific receptor on B or T cells or to an antibody. Large protein antigens usually possess multiple epitopes that bind antibodies of different specificity.
28
What is a monoclonal antibody?
An antibody produced by the progeny of a single B cell and thus a homogeneous protein that recognizes a single antigen (epitope). These can be produced experimentally using a hybridoma.
29
What is a hybridoma?
A clone of hybrid cells that are immortal and produce monoclonal antibodies. It is formed by the fusion of a normal antibody-producing B-cell with a myeloma cell.
30
What does Monastrol do?
Inhibits microtubule-based motor kinesin-5 that is necessary to separate the poles of the mitotic spindle. It is being tested as an anti-tumor drug in certain brain cancers.
31
What is the cell doctrine?
- proposed by Schleiden and Schwann in 1838 | - states that all plant and animal tissues are aggregates of individual cells
32
How is the magnification of a microscope determined?
magnification of objective lens x magnification of projection/ocular lens
33
What is resolution?
the ability to distinguish between 2 objects (minimum distance between 2 distinguishable objects) D= 0.61(wavelength)/Nsina
34
What is the resolution for simple light microscopy?
0.2 microns
35
What are the advantages of bright field microscopy?
- simple - inexpensive - no staining required - live cell imaging
36
What are the disadvantages of bright-field microscopy?
- very low contrast of most biological samples (mostly composed of water) - low apparent optical resolution (0.2 microns) due to the blur of out of focus material
37
How is a phase contrast microscope different from a bright field microscope?
- refracted and un-refracted light is recombined at the image plane to form the image - increased contrast of biological specimens - incident light passes through an annular diaphragm that focuses a circular annulus (ring) of light on the spectrum - there is a phase plate in the objective that absorbs some of the direct light and alters its phase by 1/4 of a wavelength - if a specimen refracts or diffracts the light because of the refractive index of the material, the phase of some light waves is altered - each organelle has a different refractive index which results in a more detailed image (out of phase is dark and in phase is light) - for bright field microscopy, the specimen is usually stained with dyes to enhance contrast
38
What are the steps of sample preparation for microscopy?
1. Fixation 2. Embedding (for thick specimens only) 3. Sectioning (for thick specimens only) 4. Staining
39
What happens during the fixation step of sample preparation?
- you are cross-linking the macromolecules to freeze cells and stop movement - the cross-linking agents such as glutaraldehyde and formaldehyde form covalent bonds with free amino groups - partially permeabilize cells for staining
40
What happens during the embedding step of sample preparation?
- uses wax or resins - done to fortify thick samples before they are sectioned - sample is first placed into the liquid embedding medium - then cooling or polymerization occurs
41
What happens during the sectioning step of sample preparation?
- the solid block with the specimen embedded is placed on a sectioning apparatus called a microtome - slices are cut with the knife of the microtome - sections typically cut 5 microns thick
42
What are examples of stains used in sample preparation?
- hematoxylin - eosin - Wright-Giesma - benzidine
43
What is hematoxylin?
- a blue-violet stain - basic, positively charged, cationic - binds negatively charged nucleic acids and amino acids (aspartic acid and glutamic acid) - stains the nucleus (considered a nuclear stain)
44
What is eosin?
- a pinkish red stain - acidic, negatively charged, anionic - binds basic amino acids like lysine and arginine - stains the cytoplasm proteins rich in basic amino acids
45
What is the Wright-Giesma stain?
- a histological stain for blood and bone marrow cells - a combination of eosin and methylene blue - stains the nuclei blue-violet - stains the cytoplasm pale pink - stains erythrocytes pale pink - used in differential white blood cell counts and to study red blood cell morphology
46
What is benzidine?
- stain - binds heme-containing proteins such as those seen in blood cells - used to visualize ventral blood islands (VBIs)
47
What is a fluorescent molecule?
- a molecule that absorbs light at one wavelength (excitation wavelength) and emits light at a specific longer wavelength (emission wavelength) - also called fluorophore or fluorochrome - ex: fluorescein
48
What are the excitation and emission wavelengths for fluorescin?
- excitation: 492 nm | - emission: 525 nm
49
What is the difference between fluorescence microscopy and bright field microscopy?
- in fluorescence microscopy, a dichroic mirror and excitation filter are used - the dichroic mirror acts as a filter and only emits the emission wavelength you are interested in - the excitation filter only allows the excitation wavelength you are interested in - the light source is typically a laser
50
How many colors can you see at one time on a fluorescence microscopy image?
- one at a time | - you need to merge the images to see more than one color at a time
51
What is Fura-2?
- an ion sensitive fluorescent dye - is blue in low concentrations of Ca2+ - is green in medium concentrations of Ca2+ - is yellow/orange/red in high concentrations of Ca2+ - helps to see cell motility because the release of calcium into the cytoplasm plays a role in directing motile cells
52
What is the process of using immunofluorescence microscopy to detect specific proteins in fixed cells?
1. Inject antigen X 2. Vertebrate immune system responds by generating polyclonal antibodies that recognize different epitopes on the antigen 3. Antibodies that recognize antigen X can be purified from all other antibodies and proteins in the blood serum using affinity chromatography over a resin on which the antigen is immobilized 4. Fix and permeabilize the cells that you want to test 5. Add the primary antibody that recognizes antigen X (antibody passes through the plasma membrane of the cell) 6. Add a second fluorescent antibody that is anti-(organism that the primary antibody came from) 7. The second antibody will bind to the Fc portion of the primary antibody 8. View the cells using fluorescence microscopy (portions of the cell that are fluorescent contain antigen X)
53
What is double-label fluorescence microscopy?
- allows you to visualize the relative distributions of two proteins - each protein is labeled with a different fluorochrome - still can only see one color at a time and have to merge the images to see multiple colors
54
How is an epitope tag used?
- a short peptide sequence (such as FLAG or Myc) is added to a specific gene (gene X) - transcription and translation occur to produce epitope-protein X - commercial fluorochrome-coupled monoclonal antibodies to FLAG or Myc epitopes can be used to detect the protein in the cell - tag typically does not interfere with protein's function or location - no longer need to produce a primary antibody against the protein
55
How are proteins localized in living cells?
- tag with fluorescent proteins such as green fluorescent protein (GFP) found in jellyfish - coding sequence of GFP can be fused to the coding sequence of a protein of interest - when the construct is introduced and expressed in cells, you can visualize the location of the protein of interest because it will show up as fluorescent - can track the distribution of the protein in a live cell over time
56
What is deconvolution microscopy?
- focuses on one plane at a time - computational algorithm removes fluorescence coming from different planes - focuses individually on each plane and collects optical data (fluorescence) from that plane and cancels out of focus light - images are merged using deconvolution algorithm
57
What is laser scanning confocal microscopy (LSCM)?
- makes it possible to focus on a chosen plane of a thick specimen while rejecting the light that comes from out of focus areas (confocal pinholes do this) - allows you to use thick specimens without sectioning them - preserves 3D information - much clearer than conventional fluorescence microscopy
58
What is point scanning LSCM?
- used to image "static" or "fixed" samples that don't change over time - produces a 2D image of the specimen - each and every point is recorded and merged to produce a crisp image - more time consuming
59
What is spinning disc LSCM?
- used to observe fast, dynamic events in a cell that the point-scanning microscope can't capture - light path from laser is spread to illuminate pinholes on the coupled spinning discs - as the disc spins, it rapidly illuminates all parts of the specimen several times - disc can spin as fast as 3000 rpm in order to capture very dynamic events
60
What is two-photon excitation microscopy and how does it compare to point-scanning confocal microscopy?
- in point-scanning confocal microscopy, one photon is used that has an excitation wavelength in the visible range which produces background scattering in samples over 1 mm thick - in two-photon excitation microscopy, two photons are used that have excitation wavelengths beyond the visible spectrum (infra-red, less energy) - this will not scatter light for a thick specimen - the two photons bombard the specimen at the same time and their energy adds up to double, which is enough to emit the same emission wavelength as point-scanning confocal microscopy - two-photon excitation microscopy can be used for live specimens

BIOL 4610 (7 decks)

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