flow technical

messengerrushΒιοτεχνολογία

22 Φεβ 2013 (πριν από 4 χρόνια και 7 μήνες)

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How do scientists identify types of cells?


1. Morphologically (RBC, granulocyte, lymphocyte)

2. Functionally

3. Phenotypically



Use of monoclonal antibodies directed against



cell surface proteins



Can also use certain chemicals (PNA, antigen, PI)



Involves some sort of “visible” label



radioactivity



enzyme
-
substrate reaction (histochemistry)



fluorescence

Fluorescence



Chemical or protein that absorbs light


at one wave length (excitation)

and emits light at a second wave length (emission)


First must know properties of flurochrome

you wish to use

Ethidium

PE

cis
-
Parinaric acid

Texas Red

PE
-
TR Conj.

PI

FITC

600 nm

300 nm

500 nm

700 nm

400 nm

457

350

514

610

632

488

Common

Laser

Lines

Robinson et al. Purdue Cytometry Laboratory

Propidium Iodide

400 nm

500 nm

600 nm

700 nm

Relative Intensity
PI

DNA

Excitation

Emisson

300 nm 400 nm 500 nm 600 nm 700 nm

Fluorescein (FITC)

400 nm

500 nm

600 nm

700 nm

Relative Intensity
Wavelength

Protein

Excitation

Emisson

300 nm 400 nm 500 nm 600 nm 700 nm

Excitation

Emisson

300 nm 400 nm 500 nm 600 nm 700 nm

Phycoerytherin (PE)

Protein

Allophycocyanin (APC)

Protein

632.5 nm (HeNe
)

Excitation

Emisson

300 nm 400 nm 500 nm 600 nm 700 nm

Flurochromes are generally stimulated by laser beams

(single wavelength of light).




1. Fluorescent microscope (very simple to very







complex)



tissues or single cells



2. Flow cytometer
-

single cells in suspension



Optics



Fluids



Electronics


Which to use?

Typical Clinical
Cytometer

Computer System

Detector &

Mechanical

Fluidics

Robinson et al. Purdue Cytometry Laboratory

PMT

PMT

PMT

PMT

Dichroic

Filters

Bandpass

Filters

Flow Cytometry Optics

Laser

1

2

3

4

Flow cell

Robinson et al. Purdue Cytometry Laboratory

Optical Filters

Dichroic Filter/Mirror at 45 deg

Reflected light

Transmitted Light

Light Source

Standard Band Pass Filters

Transmitted Light

White Light Source

630 nm BandPass Filter

620
-
640 nm Light

Standard Long Pass Filters

Transmitted Light

Light Source

520 nm Long Pass Filter

>520 nm
Light

Transmitted Light

Light Source

575 nm Short Pass Filter

<575 nm
Light

Standard Short Pass Filters

Fluidics
-
Flow Cell

Injector


Tip

Fluorescence

signals

Focused laser

beam

Sheath


fluid

Robinson et al. Purdue Cytometry Laboratory

Electronics



PMT collect light signal


Converted to electronic signal


Interpreted by software (Cell Quest)


Color and intensity on each cell

What can we measure
?


Light scatter


forward = size


side = granularity



Fluorescence



Time



Ratio

Robinson et al. Purdue Cytometry Laboratory

Light Scatter

FALS Sensor

90LS Sensor

Laser

Robinson et al. Purdue Cytometry Laboratory


Cell Viability


Phagocytosis


Organelle Functions


mitochondria, ER


endosomes, golgi


Oxidative reactions


superoxide


hydrogen peroxide


nitric oxide


glutathione levels


Ionic Flux


Calcium


Intracellular pH


Membrane potiential


Membrane polorization


Lipid peroxidation

Cellular Functions

Robinson et al. Purdue Cytometry Laboratory

Typical

Assays



Immunophenotyping


DNA content, cell
-
cycle analysis, ploidy


Chromosome karyotyping


Phagocytosis


Kinetic assays


Intracellular protein measurements


Total protein

Robinson et al. Purdue Cytometry Laboratory

Research uses of flow cytometery

Immunology

Hematology

Pathology

Microbiology

Genetics

Drug discovery

Toxicity testing

Cell culture studies

Functional studies


Chemical engineering

Biotechnology

Agronomy

Animal Science

Robinson et al. Purdue Cytometry Laboratory

General Protocol for Immunophenotyping


1. Make single cell suspension

2. Deplete red blood cells if necessary

3. Count cells

4. Add blocking reagent to block FC binding

5. Add dyes/mAbs

6. Incubate on ice

7. Wash out unbound dye

8. Analyze

No Fc blocking


Direct



Y

Y

Y

Y

Anti
-
CD4 FITC

=> B cells look positive!


Correct!!!

Y

Y

Y

Y

Y

Y

Y

Indirect

Biotin
-
avidin

Biotin
-
mAb

Streptavidin
-
flurochrome

Labeled 2nd antibody

1 =goat anti
-
mouse CD4

2 =sheep anti
-
goat Ig
-
FITC

Y

Y

What does flow cytometry data look like?


Histogram

Dye

Number of cells

Dye 1

Dye 2

No dye

Both dyes

Only dye 2

Only dye 1

Dot plot

488 nm laser

+

-

Fluorescence Activated

Cell Sorting

Charged Plates

Single cells sorted

into test tubes

FALS Sensor

Fluorescence detector