javad jamshidi fasa university of medical sciences, december 2015 tools and techniques for studying...

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