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CHAPTER 2
THE TECHNOLOGIES USED
IN CELLULAR BIOLOGY
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Cell culture
(1) cultured cells can be obtained in large quantity;
(2) most cultures contain only a single type of cell;
(3) many different cellular activities, including
endocytosis, cell motility, cell division, membrane
trafficking, and macromolecular synthesis, can be
studied in cell culture;
(4) cells can differentiate in culture;
(5) cultured cells respond to treatment with drugs,
hormones, growth factors, and other active
substances.
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Primary culture: The cells are obtained directly from the
organism. Usually, primary culture can be continued with limited
passage number (about 50 times).
Secondary (Passaged) culture: Cell line culture.
Tumor cells can be cultured for long time with almost unlimited
passage times.
Mass culture: Culture for tissue mass. It is not usually used
in lab.
Cloning culture: Cell culture to colonize cell. If you want to
develop some special cell line, you have to culture cell clonally.
Culture on special basement or prop stand: Cells
will grow up on some special basement or matrix.
Cell line: Cells are developed from one cell with some specific
characters.
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Basic Steps for Cell Culture
Construction of primary cell culture or cell line
Culture with medium and other help reagents
Observation and property checking
Change medium
Experimental treatments and results examination
Cell passage and cell line stock
Data analysis
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What you have to know for cell culture:
1. CO2
2. Temperature
3. pH and ion concentration
4. No any contamination
5. Start a culture from a stock
Dish
6. Passage and cell line stock
6-wells plate
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Microscopes and Images
Optical microscope
Optical inverted microscope
Fluorescence microscope
Electron microscope
Laser confocal scanning microscope
Polarized light microscope
Phase contrast microscope
Cell image station
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Simple upright light microscope
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Optical microscopes
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Fluorescence
microscope
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Laser confocal scanning microscope
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An photo taken by LCSM
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Mechanism about phase contrast microscope
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Human blood cells viewed by bright-field and phase-contrast light microscopy.
Arrow indicates a white blood cell. Formyl-met-leu-phe causes the white blood
cell to spread out and become very thin. It becomes almost invisible by brightfield microscopy but can still be detected by phase-contrast microscopy
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A photo taken by phase contrast microscope
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Optical inverted
microscope
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Transmission
Electron
Microscope
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A tissue section machine for transmission electron
microscope
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A photo about
endoplasmic
reticulum taken by
transmission
electron microscope
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Scanning Electron Microscope
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Human blood cells photo taken by scanning electron microscope
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A freeze-etching
photo about a cell
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A microscope
operation system
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Digital image microscope system
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Image station
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Powerful microscopes:
Laser confocal scanning microscope (LCSM)
1. Catches a very thin focal plane within a thick specimen
2. Image at three different planes within the specimen
without any damage to the specimen
3. Do not have to section tissue to get specimen slides for this
microscope
Scanning tunneling microscope (STM)
1. Most powerful microscope in the world so far at atom
grade
2. Stereoscopic surface image based on atomic grade
3. Specimen must be of electric conductivity
4. No any damage to the molecule’s function
Atomic force microscope (AFM)
1. Powerful also at atom grade
2. The specimen does not have to be of electric conductivity
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Histochemistry, immunochemistry,
display and other technologies
1. Isolation of the cell apparatus, biological
macromolecules and complexes:
Centrifuge
methods to
isolate cell
components
Differential
centrifugation
Density
gradient
centrifugation
If the both methods are used together, you
will obtain the better isolation result than
using one method alone.
The cell
components will
be divided
following their
different
sedimentation
coefficient, “S”
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Differential centrifugation
Using different RPM you will spin down the particles with
different sedimentation coefficient
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Density gradient centrifugation
(A) Sedimenting with same speed; (B) Sedimenting with same density
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2. Display and stain the nucleic acids, protein,
enzyme, sugar, and fat/lipid:
Nucleic acids: Feulgen reaction (Schiff reaction).
Sugar: PAS reaction.
Fatty/Lipid: Sudan III or Sudan Black reaction.
Protein: Millon reaction, Ninhydrin staining.
Enzyme: Use ubstrate reaction or the methods same to stain protein
as above.
3. Locate and quantitate the antigen in cells:
Usually, we use HRP (Horseradish peroxidase), AP (Alkaline
phosphatase), biotin or inflorescence to label the Ab against the specific Ab
against the Ag you want to check or display. We call the labeled Ab as
second Ab and the Ag binding Ab as first Ab.
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The complex of
Ag plus Ab
The labeled Ab against the first Ab
(Second Ab)
The substrate for enzyme or biotin
The cell,
bacterial,
virus, tissue
or others
with the Ag
you want to
check
Enzyme, biotin, isotope or others
The color, bright or
radio ray particles
that are visible by
your eyes or
machine
The antigen you want to
check or display from any
bio-organisms, component,
or other objects.
The Ab against
the Ag specificly
(First Ab)
Immunoenzyme methods to detect specific Ag or Ab
ELISA, ELISPOT and others
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Specific Enzymatic Detection of
Membrane-Bound Antigens
1. Unoccupied sites on the membrane
2. Primary antibody to a specific antigen
is incubated with the membrane
3. A blotting-grade antibody-enzyme
conjugate is added to bind to the
primary antibody
4. Color development reagent is added to
the blot; the HRP or AP enzyme
catalyzes the conversion of the
substrate (S) to a colored precipitate (P)
at the site of the antigen-antibody
complex
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Western blotting
Western blotting is the most important and popularly used
detection method for cell biology, molecular biology and
molecular immunology to check gene expression, antigen,
components of antigen, and antibody. The good western blotting
performance skills are the basic requirements to every body who
is working in a life science research laboratory. It is not so easy
to get good data (images).
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Basic steps for Western Blotting
– Cathode
– Cathode
+Anode
+ Anode
Develop each
band of each lane
to be visible with
a serial steps as a
image result
Protein migration direction
Gel
NC membrane
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Example results of Western Blotting
A model picture of the transfer electrophoresis of
western blotting
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A component of the Criterion precast gel system, the Criterion
blotter combines blotting efficiency and flexibility in a unit that
is incredibly easy to use
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The Mini Trans-Blot cell provides rapid, high-quality blotting of mini
gels. A component of the Mini-PROTEAN 3 electrophoresis system,
the Mini Trans-Blot cell accommodates two gel holder cassettes for
electrophoretic transfer of both mini format gels run in the MiniPROTEAN 3 cell.
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The Trans-Blot Plus cell offers rapid and effective transfers over a
large 26.5 x 28 cm blotting area. Designed for use with large format
gels, such as those used with the PROTEAN Plus Dodeca cell, this
versatile tank transfer system provides uniform transfers in as little as
15–30 minutes
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Bio-Rad
offers two
types of
blotting
apparatus
that
complement
our vertical
cells
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The Bio-Dot and Bio-Dot SF (slot format) microfiltration units provide
a reproducible method for binding protein or nucleic acid in solution
onto nitrocellulose or Zeta-Probe membrane. Many experimental
protocols can be accommodated by using interchangeable templates
to form the 96-well Bio-Dot apparatus or 48-well slot format Bio-Dot
SF apparatus. Each is available as a complete, independent unit or as
a modular template without the manifold base
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Mini incubation trays allow safe, simple, and economical screening of
antigens that have been blotted onto membranes. With these trays,
the entire Immun-Blot assay screening process can be carried out in
the tray, minimizing exposure to biohazardous materials. Because the
trays are disposable, concerns associated with washing reusable
trays are eliminated
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Using the Mini-PROTEAN II multiscreen apparatus, you can quickly and
efficiently screen up to 40 different antibodies or sera without having
to cut the western blot into individual strips. This unit clamps the blot
securely in place, creating 40 leakproof channels. Individual samples
can be screened without cross-contamination. Additionally, procedures
like screening monoclonal antibodies and monitoring antibody titers
from multiple sources are simplified
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The Model 785 vacuum blotter quickly and efficiently transfers
DNA or RNA from an agarose gel onto a nylon membrane.
Because it generally requires only 90 min for transfer, the
nucleic acid samples can be separated on a gel and transferred
to a membrane, and the hybridization reaction begun on the
same day
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Northern transfer using the Model 785 vacuum blotter. Samples
of 1.0, 2.5, 5, 10, and 20 µg of total RNA from CHO HA-1 cells
were separated on a glyoxyl gel and transferred onto a ZetaProbe membrane using 3" Hg for 90 min. The blot was probed
with a 32P-labeled hsp70 cDNA fragment* and exposed to X-ray
film overnight.
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ELISA
ELISA is always used to detect antigen or antibody in research lab or clinic
lab because it is very easy, specific, and sensitive.
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Immunofluorescence Technology
Immunofluorescence Technology was designed based on the fluorescence labeled
antibody technology. Stain the specific antigen, then you can view the stained antigen
in cells or tissue under a fluorescence microscopy or take digital images as your
experimental data.
4. Locate or quantitate the specific nucleic acid
sequence in cells or tissue:
Hybridization in situ is used for this object. Label specific oligonucleotide (DNA
probe or RNA probe) with biotin, fluorescence, or isotope, make the probe hybridized
with specific target sequence in cells or tissue, then check the samples under a
fluorescence microscopy or electric microscope.
Biotin, Isotope, or fluorescence
Oligonucleotide probe
Target DNA/RNA sequence
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Human chromosome telomeres were displayed by
Hybridization in situ with fluorescence labeled probe
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An photo taken by fluorescence microscope
Nucleus
Micro filament
Micro tubes
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5. Analyze the synthesis of the macromolecules
in cells with radio labeling technology
3H
inserting method:
1. One of the 4 dNTPs is labeled. Develop your sample as DNA/RNA image
data.
2. One of the 20 amino acids to be labeled. Develop your sample as protein
image data.
6. Flow cytometry (FCM)
1. To quantitate some molecule in or on cells
2. To check the cell subsets, immunity status
3. To sort cells into tubes for the culturing
Usually, the antibody against some special marker (first antibody) on
the cell surface is labeled by fluorescence.
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FCM
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7. Section and slides:
Paraffin section: Usually use to make slides for
long time storage
Section
Freeze section: Usually use to diagnose quickly or
keep molecule activity no changed
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Section machine
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