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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

6. Passage and cell line stock

Dish

6-wells plate

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Microscopes and ImagesOptical 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 bright-

field 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

20Scanning Electron Microscope

21Human 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 specimen2. Image at three different planes within the specimen

without any damage to the specimen3. 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

grade2. Stereoscopic surface image based on atomic grade 3. Specimen must be of electric conductivity4. No any damage to the molecule’s function

Atomic force microscope (AFM)1. Powerful also at atom grade2. 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

The cell components will

be divided following their

different sedimentation

coefficient, “S”If the both methods are used together, you will obtain the better isolation result than

using one method alone.

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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 cell, bacterial,

virus, tissue or others

with the Ag you want to

check

The antigen you want to check or display from any

bio-organisms, component, or other objects.

The Ab against the Ag specificly

(First Ab)

The labeled Ab against the first Ab

(Second Ab)

The substrate for enzyme or biotin

The complex of Ag plus Ab

Enzyme, biotin, isotope or others

The color, bright or radio ray particles that are visible by

your eyes or machine

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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+ Anode

– Cathode

+Anode– Cathode

Protein migration direction

Develop each band of each lane to be visible with a serial steps as a

image result

Gel NC membrane

Basic steps for Western Blotting

Example results of Western Blotting

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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 Mini-PROTEAN 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 Zeta-Probe 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 technology3H 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 cells2. To check the cell subsets, immunity status3. 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.

51FCM

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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