FACS Flourescens Activeted Cell s ortering

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

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

CD Cluster of diffentiation Antibody (CD)Reactivity CD1a Thymocytes and immature T cells CD2 T cells, Large granular lymphocytes (LGL), NK cells, some APL, neoplastic mast cellsLGLAPL CD3 T cells, primary effusion lymphoma CD4 T cells (helper/inducer), monocytes, myeloblasts, NK cell lymphoma CD5 T cells, B-CLL/SLL, MCLB-CLL/SLLMCL CD7 T cells, some myeloblasts CD8 T cells (suppressor/cytotoxic), large granular lymphocytes (LGL)LGL CD10 Follicle center cells, FL, some DLBCL, pre- B-ALL, pre-T-ALL, thymocytes, BLFLDLBCLpre- B-ALLpre-T-ALLBL CD11b Granulocytes, monocytes CD11c Monocytes, HCL, LGL, activated T cells, MZLHCLLGL MZL CD13 Myeloid cells, rare pre-B-ALLpre-B-ALL CD14 Monocytes

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