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93 Cards in this Set

  • Front
  • Back

RNAi

Blocks gene expression. siRNA are temporary and not carried forward with cell division. shRNA are permanent.

Tetrodotoxin (TTX)

Found in puffer fish; blocks Na+ channel in neurons = no action potential.

Saxitoxin

Red Tide

Soman/Sarin

Blocks breakdown of ACh

Ricin

Found in castor beans. Binds to ribosomes and blocks protein synthesis. When synthesized, it is proricin which is non-toxic until it leaves the cell.

Anthrax

Bacteria based. Produces an endospore. Potent. Can be stored for millions of years, resistant to UV and an inhalant.

Totipotent

Can differentiate into all types of cells. Ex: HESC



Pluripotent

Can differentiate into some cell types.

Robert Hooke

First saw "cells" in 1655.

Leeuwenhoek

First recorded (drawn) cells in 1674.

Schleioen and Schwahn

Developed Cell theory.

Cell Theory

All organisms are made of cells. Each cell is independent and is the smallest unit of life. Cells arise from other cells.

Theoretical Limit of Resolution

Abbes resolution equation, resolution cannot exceed the wavelength of visible light.

Practical Limit of Resolution

Cells have very low contrast (in light). Cells have thick sections and absorb light and heat up

Bright-Field Microscope (Compound Light Microscope)

Oldest and most used microscope.

Phase Contrast Microscope

Views Living cells. Manipulates phase shift changes as light passes through different mediums to create a clearer image.

Differential Interference Contrast

Similar to phase contrast microscopy, however it utilizes polarized light to create artificial shadows as if the specimen was lit from the side.




Used for intracellular injections and patch clamps.




Views living cells.

Darkfield Microscopy

Bright Image with dark background. Condenses light on cells.



Polarized Microscopes

Use polarized light. Highly ordered parallel structures that change the orientation of light.

Confocal Microscope

First made in 1988. Increased practical limit by imaging cells spot by spot and combining into one 3D image.

Fluorescence Microscopy

Fluorescence dues used to identify molecules and examine changes in cell physiology, gene expression.

Acetoxymethyl ester (AM)

Attaches to fluorochromes to make them permeable to the cell membrane.

Fluorescence Recovery After Photobleaching (FRAP)

Measures membrane mobility - which proteins move within lipid bilayer.




Mark membrane proteins, then photobleach section with laser, then watch mobile proteins move.

Fluorescence Immuocytochemistry

Locates cellular molecules using fluorochromes and antibodies.

Polyclonal Antibody

Non-specific binding sites, easy to produce in large numbers and recognizes multiple epitopes on any one antigen.

Monoclonal Antibody

Single clone, single antibody.




Immortal - unlimited source.




Made by fusing B cells and lymphoma B cells

ELISA - Enzyme-linked Immunosorbent Assay

Measures amount of antigen (quantitative).




Antigens from a sample are attached to the surface of the plate. Then an antibody linked to an enzyme bind to the antigens. When the enzymes substrate is added, the color change shows the amount of antigen present in a sample.

Propidium Iodide

Binds to nucleic acids. Allows visualization of DNA and RNA.

Annexin V

Measures apoptosis.




Binds to phospahtidyl serine of cell membranes. This molecule is usually on the inside of the cell until it begins apoptosis.

Green Fluorescent Protein (GFP)

Reporter molecule. Can be attached to gene of interest and observe where it is in the cell.

Fluorescence Resonance Energy Transfer

Measures protein-protein binding and ligand receptor interactions.




When one molecule is excited and releases a photon of the excitation wavelength of a molecule next to it, it then excites this neighbor.

Autoradiography

Tracks radioactive probes. Examples:




DNA Synthesis: Add 3H-thymidine to medium and any cell undergoing DNA synthesis will emit dots in the nucleus.




RNA Synthesis: Add

In situ hybridization

Search for presence of genes or expression of select mRNA.




Can be used to locate the location of specific nucleic sequences in the cell.

Micropipet Intracellular Injection

Can inject protein, nucleus, etc.




Only 1 micrometer opening and can only do one cell at a time.

Electroporation

Add voltage to many cells to open cell membrane pore. Very brutal to cells.

Liposome fusion

Standard transfection technique. Uses liposomes that fuse with the membrane and release substance into the cell.

Gold particle

Used with DNA and RNA.

Transmission Electron Microscope (TEM)

Shoots electrons through specimen to produce image on other side.




Can view internal structures.

Scanning Electron Microscope (SEM)

Views surfaces of specimens. Lower resolution than TEM.

Cutting thin sections

Fix tissue with OsO4 and glutaraldehyde; dehydrate, cut ultra-thin sections; stain with metal then ready for TEM.



Freeze Fracture

Allows you to view internal structures of membranes; fracture follows line of least resistance. Stain with platinum.

Ultrastructural Immunochemistry

Use antibodies to localize proteins in cells.




Attach gold particle to antibody; double label with two different sized gold particles.

Electron Tomography

Computer reconstructs 3D image

Laser Capture Microscopy

Identify cells of interest and dissect them for further analysis. Laser removes film on top of cells.

NMR microscopy

Can view living tissue. Low resolution.

Atomic Force Microscope

No lenses involved and involves fine tip. Can view DNA and cell pores.

Fluorescence Activated Cell Sorting (FACS)

Separates living cells tagged with specific fluorescent dye.

Selective Surfaces

Rely on the ability of protein A to bind to ligand A.




Coat culture dish with specific ligand and only one cell type will stick to the plate.



Magnetic Beads (Dynabeads)

Beads have iron core and are coated with antibodies so cells stick to them. Can be removed after with a magnet.

Cell fractionation

Sonicate cells - destroy all membranes


Detergent - solubilize all membranes


Trituration - mechanism disruption



Ion exchange chromatography

Separates proteins based on their charges.


Column w/ buffer that contains beads.

DEAE beads

Most common b/c most proteins have overall negative charge.




Positive charge on outside.

CM

Negatively charged beads

Fast protein liquid chromatography

Uses computers, pressure pumps and improved columns.

Gel Filtration

Separates proteins based on size. Larger proteins will come out faster.

Affinity Chromatography

Purifies proteins based on ligand-receptor interaction.

SDS gel electrophoresis

SDS = detergent; Urea = disrupts H-bonds; B-mercaptoethanol = disrupts disulfide bonds.




Much higher resolution and polymetric nature of proteins. Linearizes proteins.

Isoelectric Focusing

Proteins are separated by isoelectric points.

Western Blot

Combination technique of gel electro and "blotting".




1). Separate proteins in gel. 2) Blot proteins onto membrane. 3) Block membrane to prevent non-specific binding. 4) Add labeled antibody.

Cell Strain

Cells isolated from fresh tissue, but limited to the Hayflick limit.

Cell Line

Cells are immortal.

Serum-supplemented media

From fetal/newborn bovine.



Defined medium

No serum, needed for cell therapy and decrease in cost.

Growth Substrate

Cell culture plates.


Microcarriers = beads


Roller bottles


Tubes


Microporous membrane

In vivo

In living

In vitro

In glass

In situ

In self

Self-induced Mutation

UV Light


Ethylmethane sulfonate - permanently changes C-G to A-T

Point Mutation

Single Base modification

Silent Mutation

No effect caused by mutation

Missense mutation

Change in phenotype

Southern Blot

Patterned after Western Blots.




Separate DNA on DNA gel - then transfer to paper - then use DNA/RNA probe to detect presence of a particular gene.




Both qualitative and quantitative.

VNTR

Shows genetic polymorphism.




Often used in forensic medicine.

Northern Blots

Shows gene expression by analyzing mRNAs with probe.

cDNA Microarrays

Relies on nucleic acid hybridization.




Use revere transcriptase on mRNAs from cell and make cDNAs with radioactive tags.

Cryostat

Device to maintain sample at low temperatures for extended periods of time.

Hematoxylin and eosin stain

Hematoxylin (purple) stains negatively charged cellular components, principally the nucleus. Eosin (pink) stains positively charged molecules, principally the cytoplasm.




Best used under bright-field microscopy.

Spinning Disk Confocal Microscope

Especially suited for analyzing dynamic events in living cells that occur during short periods of time.

Laser scanning Confocal Microscopy

Laser scans entire surface of specimen and creates 3-d image with depth.

Spectrofluorometer

Measures the quantity of desired analyte based on amount of fluorescent probes.

Two-photon Microscopy

Use of red-shifted excitation light to view fluorescent probes on thin layers of cells. Two photons are absorbed as IR light and this minimizes scattering within the tissue.




Minimizes photocytoxicity, deeper tissue penetration and efficient light detection.

Calcein-AM

Used to measure cell viability. Dead cells do not remove the AM portion, thus only live cells are stained green.

Fluo-3 and Fura-2

Calcium indicators.

Sirtuins and Resveratol

Molecules and medicines that may influence cellular and organismic longevity.

Quark Pharmaceuticals and Merck

Using RNAi's to tackle diseases of the eye.

Jackson Laboratory

Famous lab in Bar Harbor, Maine that has a large collection of transgenic mice.

Secretome

The totality of secreted organic molecules and inorganic elements by biological cells and tissues.




Consisted of Proteins.

Heterokaryon

Multi-nucleate cells

Hybridomas

Hybrid cell used as the basis for the production of antibodies in large amounts

Cameleon Protein

First developed by Robert Tsien, and designed to indicate changes in intracellular calcium.




Relies on FRET

Feeder Layer Cell Culture

Excellent at growing hESCs

Reticular Theory

Theory that states that the entire nervous system, including the brain, is one single continuous network.

Madin-Darby Canine Kidney epithelial cells (MDCK)

Accepted at the general model for epithelial cells.