Analyzing Cells Flashcards
SEM
(scanning EM): produces an image of the 3D structure of the surface of a specimen
Electron Microscopy
electron beam (not light)
TEM allows
(transmission EM): allows visualization of internal cell structures
Limit of resolution of TEM
much smaller than light microscopy, about 200x better at 1 nm
TEM is similar to light microscope, only ___
upside down and larger
Electron Source in TEM
Cathode and anode
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
TEM imaging
Some electrons passing through specimen are scattered (by electron dense stained structures), remainder focused to form an image.
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
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
osmium tetroxide
preserve lipid bilayers
formaldehyde
free ameno group is locked into place
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)
Scanning electron microscope produces __
Preperation __
- Directly produces image of 3D structure of the specimen surface
- Specimen is fixed, dehydrated, coated with heavy metal.
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
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)
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
FACS
point/used to __
Point - to have populaton of cells to studdy (not killed)
Used to separate a cell type from mixed suspension
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
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
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
- Spleen cells removed (B cells): produce antibody, has enzyme
- B cells fused with Myeloma Cells (Cancerous B cells - immortal) to make a hybridoma, not have enzyme
- 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).
