FACS Flourescens Activeted Cell s ortering. användningsområden.
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Transcript of FACS Flourescens Activeted Cell s ortering. användningsområden.
FACS Flourescens Activeted Cell sortering
användningsområden
Measurable parameters in flow cytometry
•volume and morphological complexity of cells •cell pigments •DNA (cell cycle analysis, cell kinetics, proliferation etc.) •RNA •chromosome analysis and sorting (library construction, chromosome paint) •proteins •cell surface antigens (CD markers) •intracellular antigens (various cytokines, secondary mediators etc.) •nuclear antigens •enzymatic activity •pH, intracellular ionized calcium, magnesium, membrane potential •membrane fluidity •apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes) •cell viability •monitoring electropermeabilization of cells •oxidative burst •characterising multi-drug resistance (MDR) in cancer cells •glutathione •various combinations (DNA / surface antigens etc.)
FACS model
Hydro-dynamic focusing
laminärntflöde
Tubulent flöde
Laminärtflöde parametrar
Light scattering – size of particle
Scattering
Forward scatter
Side scatter
http://www.epa.gov/owow/volunteer/proceedings/sixth/session1.html
Side scatter vs Forward scatter
University of Utah
scatterplot
Fluorimeter
fluorimeteroptik
Vaccine Research Center / 40 Convent Drive / Bethesda, Maryland 20892
FACS diagram
Fluorescens plott
Lin och log
Flourescens colours
Fluorescein
Sample : peripheral blood after red cell lysis.Data collected on 10,000 cells.
CD45 FITC (log)
R1=monocytes(CD14+ve) R2=lymphocytes (CD45>monocytes)R3=granulocytes (CD45<monocytes)
CD14
PE (lo
g)
Forward Scatter (linear)
Sid
e Sca
tter
(lin
ear)
Sample : peripheral blood after red cell lysis.Data collected on 10,000
Scatter
Low
High
Events
EventsCD14 PE
CD 45 FITC
Fluorescence
Isometric Displays
Apoptotic cells
As cells die or become apoptotic the refractive index of the internal cytoplasm becomes more similar to that of the extracellular medium - this manifests itself as a reduction in forward scatter signal. At the same time, intracellular changes and invagination of the cytoplasmic membrane lead to an increase in side (or orthogonal or 90°) scatter. If we add a dead cell discriminatory dye we can identify cells that have become permeable. In this way we can get low level resolution of dead and apoptotic cells.
science.cancerresearchuk.org/.../images/51998
endotelceller
Isolation of lymph endothelial cells (LEC) from dermal skin capillary ECs
a case study with cells from one donor pre-selected by CD31 magneto bead
sorting
R2= total cells R1= living cells
www.reliatech.de