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J a v a d J a m s h i d i
F a s a U n i v e r s i t y o f M e d i c a l S c i e n c e s , D e c e m b e r 2 0 1 5
Tools and
Techniques for Studying Cells
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Outlines
Cell Culture
Flow Cytometry
Microscopy
Centrifugation
Gel Electrophoresis
Blotting
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Study cellsIn 1655, Robert Hooke used a primitive microscope and coined the term cells.
As study of cells in intact animals and plants is difficult, molecular cell biologists often conduct experimental studies on cells isolated from an organism
Culture of unicellular bacterial, fungal, or protist cells is easier than animal cells come from multicellular organisms
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Animal Cells Culture Requirements
Closely as possible the conditions within an intact organism
TemperaturepH Ionic strengthAccess to essential nutrients The nine amino acids (phe, val, thr, trp, ile, met, leu, lys, his)
Antibiotics
Most animal cell types will grow only attached to a solid surface
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Primary Cell CulturesTissue fragments are treated with a combination of a protease (e.g., trypsin, collagenase, or both) and a divalent cation chelator
The released cells are then placed in dishes in a nutrient-rich, serum- supplemented medium
Fibroblasts usually divide more rapidly than other cells
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Stages in the establishment of a cell culture.
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Cell LinesCells derived from some tumors, cell line
The Hela cell line, the first human cell line established, was originally obtained in 1952 from a malignant tumor (carcinoma) of the uterine cervix
Cells in immortalized lines often have chromosomes with abnormal DNA sequences
Growth of Cells in Two-Dimensional and Three-Dimensional Culture Mimics the In Vivo Environment
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Flow Cytometry
Flow ~ cells in motion
Cyto ~ cell
Metry ~ measure
Measuring properties of cells while in a fluid
stream
Measures cell properties and sorting them
FACS
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Microscopy
Light microscopy: uses visible light to observe specimens
Bright-field microscopy
Dark-field microscopy
Phase-contrast microscopy
Fluorescence microscopy
Electron Microscopy: Uses electrons instead of lightTransmission electron microscope (TEM)
Scanning Electron Microscopy (SEM)
A sense of scale betweenliving cells and atoms
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Fluorescence MicroscopyPerhaps the most versatile and powerful technique for localizing molecules within a cell by light microscopy
Uses UV light and florescence dye
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Electron Microscopy
Uses electrons instead of light
The shorter wavelength of electrons gives greater
resolution
The limit of resolution is theoretically 0.005 nm
The effective resolution in the study of biological systems is
0.1 nm, still about 2000 times better than with
conventional high-resolution light microscopes
Transmission Electron Microscope (TEM)
Scanning Electron Microscopy (SEM)
The principal features of alight microscope and a transmissionelectron microscope.
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Scanning Electron Microscopy (SEM)
Scanning Electron Microscopy (SEM)
Scanning Electron Microscopy (SEM)
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CentrifugationThe principle behind centrifugation is that two particles in suspension with different densities will settle at different rates
Centrifugation is used for two basic purposes: As a technique to separate one type of material from others As an analytical technique to measure physical properties (e.g., molecular weight, density, shape) of macromolecules.
The sedimentation constant, s, is a measure of sedimentation rate.
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Types of Centrifuges
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Centrifuges Rotors
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Centrifuge Blood
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Gel Electrophoresis
An analytical method based on movement of charged particles because of an external electric field
To separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments
Velocity of a particle depends on the: size, shape and charge of the particlegiven applied voltage
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Types of GelAgarose
Are easily cast and handledAre typically run horizontally DNA fragments ranging from 50 base pair to several megabases
Polyacrylamide Handling is more difficultAre run in a vertical configurationHigher resolving power for DNA 1bp
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Types of Gel
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Direction of Electrophoresis
Horizontal ElectrophoresisVertical Electrophoresis
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Direction of Electrophoresis
Horizontal ElectrophoresisVertical Electrophoresis
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Blotting
Is a method of transferring proteins, DNA or RNA, onto a carrier
Southern blot for DNA
Northern blot for RNA
Western blot for Proteins
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Blotting
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