1. more homogenious population of cells 2. Conviencenc (easier than making animal populations) 3. Given approprate surroundings,most plant and animal cells live, muliply, and express diffenterated properties in a culture dish

Analyzing Cells Flashcards by Amelia Harman

SEM

(scanning EM): produces an image of the 3D structure of the surface of a specimen

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Electron Microscopy

electron beam (not light)

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TEM allows

(transmission EM): allows visualization of internal cell structures

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Limit of resolution of TEM

much smaller than light microscopy, about 200x better at 1 nm

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TEM is similar to light microscope, only ___

upside down and larger

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Electron Source in TEM

Cathode and anode

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How TEM Works

Cathode is a tungsten filament which emits electrons when being heated.

The beam is then accelerated towards the specimen by the positive anode.

Electron beam travels down column, magnetic coils placed in column to help focus beam. similar to a glass lense

Air is bing pumted out (via vaccume) because it (air) can colide with elctrons, causing them to scatter

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TEM imaging

Some electrons passing through specimen are scattered (by electron dense stained structures), remainder focused to form an image.

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Would you expect dense regions of the specimen to show up as dark or light areas?

if electron dense area/region - electrons are repelled by e density - darker - because electrons are scattered/not focoused

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TEM Sample Preparation

Overall - much more harsh

Fixation - perseve sample as close to native state as possible (ex. osmium tetroxide, formaldehyde)
Dehydration - presence of water would cause sample to colapse under the vacume
Embed Sample - permeate with resin/solid block of plastic so can cut into thin sections
Cut into thin sections - has to be very thin, because electrons have limited pentrating power
Stain: Heavy metal - differnt electron desnity , better contrast
Label specific proteins(?) Immunogold - conguate antibody with gold - shows up as dark dot - still specific targeting

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osmium tetroxide

preserve lipid bilayers

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formaldehyde

free ameno group is locked into place

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TEM 3D reconstruction

Thin sections (cut by microtone) often fail to convey 3D arrangement of cellular structures

View specimen from different directions by tilting specimen holder (in CT scans: imaging equipment is moved around patient)

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Scanning electron microscope produces __

Preperation __

Directly produces image of 3D structure of the specimen surface
Specimen is fixed, dehydrated, coated with heavy metal.

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SEM Pros

•Smaller, cheaper than TEM. Uses electrons that are scattered or emitted from specimen.

provides great depth of field (image has highlights and shadows that give 3D appearance).

Can view all structral details without cutting

great surface detail

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Biochemical analysis

Dissociate cells from tissue and separate them according to type

Disrupt extracellular matrix (connective tissue) and cell-cell junctions (or cell adhesions)
Separate different cell types from mixed cell suspension (Fluorescence-Activated Cell Sorter, FACS)

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How to Disrupt extracellular matrix (connective tissue) and cell-cell junctions (or cell adhesions)

Proteases

Ca kelators - bind to Ca+ and prevent from being capable of making cell adhesions

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FACS

point/used to __

Point - to have populaton of cells to studdy (not killed)

Used to separate a cell type from mixed suspension

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FACS process

Cells-add fluorescently labeled Antibody (specific for that cell type)

Sheth fluuid - differnt flow rate

Cells travel in fine stream, pass through laser (fluorescence detected)

Vibrating nozzle, tiny droplets form (one or no cell) - pass single file

Inegration point - where analized by lazer

At moment of droplet formation, charges assigned to cells (dependent on cell fluorescence)

Cells deflected by electric field, collected

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in vitro benifits

more homogenious population of cells
Conviencenc (easier than making animal populations)
Given approprate surroundings,most plant and animal cells live, muliply, and express diffenterated properties in a culture dish

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Animal cells Require ___ for growth

solid support

(ie plastic culture dish)

Proliferation of cells typically involves ___

coating dish with material cells adhere to (ie Extracellular Matrix components)

Primary culture is

Prepared directly from tissue of an organism

Secondary culture

Cells in primary culture are induced to proliferate

Passaging of cells - take small amount and put in dish with new media

Cell lines are most easily generated by \_\_

cancer cells (transformed cell lines): Indefinite replication in culture

Transformed cell lines-can

proliferate to a much higher density in culture dish combine to devide indefinatly nromal cells have contact inhibiton - cancer cells do not - grow into higher densities

HeLa cells came from \_\_ are worlds first \_\_ used to \_\_

Henrietta Lacks-Diagnosed with cervical cancer. At Johns Hopkins-George and Margaret Gey were successful in culturing **her cancer cells (HeLa), the world’s first immortal human cell line** 1954, Jonas Salk developed a vaccine for polio using these cells. HeLa cells have been used to investigate cancer, viral growth, protein synthesis, effects of radiation on cells. They have traveled to space for experiments!

Forward scatter in FACS

Size

Side Scatter in FACS

Shape and cell complexity

How Hybridoma Cell lines-Produce Monoclonal Antibodies

1. Spleen cells removed (B cells): produce antibody, has enzyme 2. B cells fused with Myeloma Cells (Cancerous B cells - immortal) to make a hybridoma, not have enzyme 3. HAT medium: Selective media for hybridoma cells. (Blocks de novo DNA synthesis, other precursors allow an alternate “Salvage” pathway, if cell has the right enzymes.)

Unfused B cells

has enzyme, finite lifespan

Unfused myeloma cells

does not have enzyme

What happens to telomeres (ends of chromosomes) after cell division

They shorten – leading to short lifespan of many cells

Herceptin targets

cancer cells that “overexpress,” or make too much of, a protein called HER–2, which is found on the surface of some cancer cells.

Herceptin attaches to

the HER–2 (receptor) positive cancer cells and slows or stops the growth of the cells. only used to treat HER-2 positive breast cancers

% of breast cancers that overexpress HER-2`

20-30%

Herceptin works by

attaching to HER2 recptor causing - immuno response - blocking intercllular signling (blocking proliforation) enhances chemotherapty (chemo drugs can aslo be attached to the monoclonal antiboty)

Cell Fractionation

Cells can be separated into their component fractions Osmotic shock (change fluid and electrolite balance), ultrasonic vibration, forced through small orifice, blender

Plasma mebrane and ER membrane in Cell Fractionation

(break into fragments that reseal to form vesicles)-retain biochemical properties

left intact in cell fractionation

Most organelles (ie nucleus, mitochondria, etc) left intact

Suspension of cells in cell fratonation is called

Homogenate (or extract): Contains organelles with a distinct size, charge, and density

Cell fractionation components/ speeds

Low speed Cell fractionation

pellet contains who cells nuclei cytoskeletons

Medium speed Cell fractionation

pellet contians mitochondrea lysosomes peroxisomes

High speed Cell fractionation

pellet contains microsomes small vesicles

Very high speed Cell fractionation

pellet contains ribosomes viruses large macromolcules

What is the advantage of using monoclonal antibodies compared with polyclonal antibodies?

The process used to produce the antibodies provides unlimited supplies NOT: They are less labor intensive to generate. They can recognize more than one epitope They can be generated without using animals.

Polyclonal vs monoclonal antibodies

Why is EDTA added in the isolation of cells from tissues?

EDTA binds to calcium, which disrupts cell-cell junctions

why is heavy metal staining is used to improve contrast of biological tissues examined by electron microscope

biological tissues are composed mainly of atoms of very low atomic number (carbon, nitrogen, oxygen, hydrogen). To make them visible, tissues are stained with electron-dense material (ie metal salts).