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

hoy@cf.ac.uk

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

hoy@cf.ac.uk

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