Intro to Cell Biology Flashcards
Cells are supplied with
- Proper nutrients
- Serum
- Grown at 37C in CO2 Incubator
Primary Cell Culture
- Divide limited number of times
- Contact Inhibition
Cell Line
- Transformed Cells (HeLa)
- Immortal
- Lose Contact Inhibition
Morphology Primary vs Transformed
Primary
- elongated + ordered
Transformed
- rounded + disorganized
Symmetric Cell Division
- divides into 2 identical cells from originating
Asymmetric Cell Division
- important for?
- divides into 2 cells uniquely different from originating
- divides into 2 cells, one is same as originating, one different
- Stem Cell Renewal
Progenitor Cells
- give rise to differentiated cells
Embryonic Stem Cells
- induced to differentiate into precursors for various cell types
- pluripotent
Embryonic Stem Cells Procedure
- remove inner cell mass from blastocysts
- culture ICM on fibroblast feeder cells
- dissociate surviving ES cells
- Plate ES cells on new feeder cells
- allow colonies to form
Adult Stem Cells
- limited differentially
- located in stem-cell niche
- adjacent cells provide signals to self-renew or differentiate
Induced Pluripotent Stem Cells
- differentiated cells with Yamanaka Factors
- Oct4, Sox2, Klf4 = embryonic stem cells
- c-Myc = cancer cells
Anton van Leeuwenhoek Microscope
- grinding lenses
- naturally acute eyesight
- 200x magnification
Bright-Field Microscopy
- light source
- condenser, objective, ocular lens
- only high refractive index
Resolution
- ability distinguish between 2 very closely positioned objects as separate objects
- smaller resolution = better
Resolution Equation
D = (0.61 λ)/(Nsinα)
Certain parts of the cell ____ than other parts.
Waves out of phase results in ___ and ____.
Cells with __ refractive properties can ___ passage of a light beam by ___.
- refract more light
- interference and dim light
- high slow 1/4λ
Phase Contrast Microscopy
- live “unstained” cells
- annular diaphragm and phase plate
- 1/2λ reduction – darker contrast
- phase halo
Differential Interference Contrast (DIC) Microscopy
- live “unstained cells”
- use polarized light
- defines outline and detail of cell (3D look)
Fluorescence Microscopy
- emit visible light when absorb light at specific wavelength
- excited with fluorochromes
- instability drops electron releasing energy
Fluorescence Microscopy Microscope
- excitation filter – selects wavelength
- dichroic mirror – reflects excitation and passage of emission light
- cell specimen labelled with fluorescent dyes that have SPECIFIC excitation/emission wavelengths
Fluorochromes
- allow labelling of more than one protein at same time
- DAPI = blue
- Mitotracker = red
Monoclonal Antibodies
- inject mouse with antigen X = antibodies in spleen
- fuse spleen cells with myeloma cells
- transfer to HAT medium
- unfused cells die (myeloma toxic to HAT, spleen reach Hayflick limit)
- fused cells are IMMORTAL
Immunofluorescence Microscopy
- kill + freeze structures
- primary antibodies bind to specific protein
- secondary antibodies (with fluorochrome) bind to primary
Fluorescent Imaging in LIVE Cells
- Green Fluorescent Protein
- excited with blue light = emits green
- fused to gene of interest on plasmid + transfected
Confocal Microscope
- laser excite point of focus
- only excited in focal plane pass through pinhole
Deconvolution Microscopy
- math procedure remove fluorescence from out-of-focus parts
- images take at different focal planes = Z-stack
- images restored by deconvolution (sharp details)
Two-Photon Excitation Microscopy
- two pulsed excitation photons at long wavelength
- image thicker samples
Fluorescence Resonance Energy Transfer (FRET)
- 440nm –> 480nm (blue) no protein interaction
- 440nm –> 535nm (yellow) protein interaction
- energy from first protein excites the second protein in close proximity
Electron Microscopy
- fixed and sectioned samples (NOT LIVE CELLS)
- specimen stained with heavy metals
- light –> electron towards anode –> magnetic condenser focuses electrons on specimen
Transmission vs Scattered Electron Microscopy
Transmission
- thin sections of cells (intracellular structure)
- electrons hits specimen, deflect due to heavy metals
Scattered
- intact cells
- angle electron beam hits (3D structure)
Immunoelectron Microscopy
- primary antibody bind to protein of interest
- gold particle linked to Protein A bind to primary antibody
- electron dense spots
