Principles of Flow Cytometry (´FACS´-Analysis) · Flow cytometers gather information about cells...

PrinciplesPrinciples of of FlowFlow CytometryCytometry

((´́FACSFACS´́--Analysis)Analysis)(FACS: fluorescence activated cell SORTING)

Fall Semester 2010/2011

Ernst MüllnerMFPL (Max F Perutz Laboratories)Department für Medizinische Biochemie

[email protected]/index.php?cid=79(Müllner homepage / research)

Allows a single cell to be measured for a variety of characteristics as they flow in liquid. Flow cytometers gather information about cells by measuring visible and/or fluorescent light emissions. This allows to sort cells based on physical, biochemical and antigenic traits. Flow cytometry is used for applications in immunology, pathology and medicine, and basic research. Most common APPLICATIONS by relevance

Evaluation of Cell-Surface Markers: determine the types of markers and receptors on the surface of a cell. A fluorescent dye is attached to antibodies or receptor ligands, cells can then be subjected to flow cytometry and the amount of the receptor on their surface detected as a level of fluorescence. Possibility to detect multiple cell-surface markers simultaneously.

Cell Sorting: Select and purify a specific subset of cells within a population. You can select cells expressing a particular receptor, in a phase of the cell cycle, or perhaps expressing a particular transgenic protein, followed by culturing a pure population. A florescenc activated cell sorter (FACS) can sort up to 30,000 cells/sec to a very high purity (over 98%).

Determination of DNA Content: Fluorescence staining of DNA followed by flow analysis to determine a cell's DNA content. Cells with one copy of their genetic material (haploid) will be half as bright as cells with two copies (diploid). A cell varies between these states during the cell cycle, thus flow cytometry can be used to determine its position in the cell cycle based on its DNA content.

Evaluation of intracellular protein abundance: cells are fixated and permeabilized by specific protocols before staining. Still in early stages of implementation but large potential.

Flow cytometry: A technology to COUNT and SORT cells

Ernst Müllner, 2010-11

below:basic components of a flow cytometer: fluorescence is detected at an angle from the emission source, i.e. at a lower wavelength of less energy.

right:basic principle of cell sorting by a flow cytometer

Flow Cytometry: The instrument I


The instrument II - light - path / common fluorochromes / filter set in a typical flow cytometerat the example of a FacsCalibur (Becton Dickinson) used in the course

Despite the use of ‘only’ two laser light sources, several wavel-engths can be detected simul-taneously by appropriate choice of dyes and filters


Fluorochrome specification


Principles of multi-colour / multi-parameter measurements Idifficulty of data representation in more than 3 dimensions

Even three para-meters only can be properly shown by animation of a diagram like on the left.

But up to eight colors can be detected simultaneously


Principles of measurement / data presentation IIExample: scheme of CD4 and CD8 T lymphocytes

representation as 2-D dot-plot representation as 1-D histogram2 parameters (CD4 + CD8) (e.g. of CD4 alone); A = background

cell number per channelErnst Müllner, 2010-11

Possibilities of presentation IIIa - ‘Real’ example in 2D: Cell-surface expression of transferrin receptor-1 is strongly reduced in Stat5-/- erythroblastsKerenyi, M. A., Grebien, F., Gehart, H., Schifrer, M., Artaker, M., Kovacic, B., Beug, H., Moriggl, R., and Mullner, E. W. (2008), Stat5 regulates cellular iron uptake of erythroid cells via IRP-2 and TfR-1. BLOOD 112, 3878-3888.

Flow cytometry histograms of E13.5 wt and Stat5-/- fetal liver cells stained for the erythroid markers TfR-1 (transferrin receptor; iron import) and Ter-119. The sequence from gate R1 (TfR-1low / Ter-119low) to gate R5 (TfR-1low / Ter-119high) represents development from the most immature erythroid progenitors (late BFU-E; CFU-E) to mature erythroid cells (orthochromatic erythroblasts, reticulocytes).

Cell-surface expression of TfR-1 of Ter-119high-gated wt (dark gray line) or Stat5-/- (light gray line) fetal liver cells (left). Quantification of TfR-1 cell-surface expression of wt and Stat5-/-

fetal livers (right; data are presented as means SD; n 4).

Overlay of several individual 1-D data sets

Corresponds almost exactly to course experiment

Marker for apoptosis = Annexin V (inside out in dying cells)Bcl-xL anti-apoptotic proteinMcl-1 anti-apoptotic protein you use Bcl-2 and DNA-profiles

Possibilities of presentation IV


colour-coding of 2D data 3 parameters; additional gating up to 5-D plots

Possibilities of presentation V


contour-coding of 2D data 3 parameters; additional gating result shown (3-D)

Possibilities of presentation VI


CMP: common myeloid precursor; CLP: common lymphoid precursor; GMP: granulocyte / macrophage progenitor, MEP: megakaryocyte / erythroid progenitor

A simplified scheme of hematopoiesis

A frequent application of flowcytometry:

characterise normal and/orabnormal blood cellpopulations from patients

lymphoidcells

myeloidcells


Functions of mature blood cell types

Granulocytes

Neutrophils respond to chemotactic signals by emigration between endothelial cells (extravasation). Mediators of this process include substances produced by microbes , and by cells participating in the inflammatory process (e.g. interleukin-1 released by macrophages as result of infection or tissue injury, or histamine, released e.g. by circulating basophils). Neutrophils support the killing of phagocytosed bacteria.

Eosinophils give large parasites a hard time. Upon activation (via C3b) the cells release various substances from their eosinophilic granules (include peroxidase, arylsulphatase, phospholipase and histaminase etc). These enzymes are capable of damaging parasite membranes.

Basophils are non-phagocytic which upon activationalso release numerous compounds from basophilic granules withintheir cytoplasm. They play a major role in allergic responses.

Platelets are no cells but fragments of megakaryocytes (residing in the bone marrow) released into the blood stream.Upon injury, platelets gather and stick to the edges of the wound. They release chemicals that help start the process of blood clotting so that bleeding will stop.


Zellen in humanem Blut

Normale Erwachsene

Zelltyp % abs/microl

Neutrophile G., stabkernig 3-5 120-450Neutrophile G., segmentkernig 50-70 2000-6300Eosinophile Granulozyten 2-4 80-360Basophile Granulozyten 0-1 50Lymphozyten 25-40 1000-3600Monozyten 2-6 80-590

Lymphocytes respond to presented antigens by production of antibodies (B cells), or lymphokines (T and B cells). Lymphocytes control of the adaptive immune response by secondary action on the participating cells, and, in the case of cytolytic T cells, kill virally-infected host cells.

Monocytes circulate in the peripheral blood prior to emigration into tissues. Within certain organs, they have special names, e.g. in liver they are known as Kupfercells, in brain as microglia, in kidney as mesangial cells, and in bone as osteoclasts (!). Elsewhere they are referred to as tissue macrophages.


Flow cytometry: example of a differential set-up for characterisation of hematopoietic cellscf St Anna Childrens Hospital

FITC PE PC5 ECD discrimination between

CD15/45 CD56 CD14 CD3 granulocytes / NK cells/ monocytes / T cells 1

CD15/45 CD8 CD4 CD3 granulocytes / cytotoxic T cells / T helper cells / T cells 2

CD15 CD33 CD34 CD45 granulocytes / pluripotent / myeloid progenitors 3

CD19 CD45RA CD34 CD45 B cells / pluripotent progenitors 4

CD15/45 CD38 CD34 CD3 granulocytes / pluripotent / lymphoid progenitors 5

GPA CD71 – – erythoid lineage 6

CD116 CD117 – – monocytic / erythroid cells 7


1 CD15 positive signals combined with CD45 high indicates granulocytes; CD56 positivecorresponds to natural killer (NK) cells; CD14 is a specific marker for monocytes and CD3is found on all mature T cells.

2 CD3 versus CD4 versus CD8 allows to discriminate between sub-populations of T cells:CD4/CD8 double positive: developing thymocyte, CD4 positive: helper T cells, CD8:cytotoxic T cells.

3 CD33 is found on myeloid committed progenitors, CD34 in combination with CD45dim is ahallmark for early (potentially pluri-potent) progenitors.

4 CD19 plus CD45 is characteristic for mature B cells; CD45RA positive signals confirm thisclassification.

5 CD38 is found on lymphoid progenitors, these cells in consequence should be CD3negative; CD34 in combination with other markers (see notes 3 and 4) is needed to derivemean values of CD34 positive and lineage-restricted negative populations in variouscompartments.

6 To indicate erythroid commitment, GPA high can be used in conjunction with hightransferrin receptor expression (CD71), since by itself its background may be too high.

7 CD116 is found on immature monocytes; the combination with c-Kit (CD117; erythroidspecific) serves no particular discrimination and just allows to measure these two cell typessimultaneously.


realistic view of a blood SmearMay Giemsa stainmagnification 1000 x

Description of the CD-antibodies used

anti-CD14 (ECD), a marker for monocytes will be combined with

anti-CD3 (PE-labeled), a marker for all T cells anti-CD34 (PE-labeled), an early progenitor marker (not really

stem cell specific) is always stained in combination with CD45, to exclude false positive cells. CD34+ cells are CD45dim (= weak positive) anti-CD45 (FITC-labeled), maturing/mature myeloid cells


Flow Cytometry: Distribution of mature mononuclear cellpopulations in human umbilical cord blood

CD3+ CD15+CD19+ CD56+

0 256 512 768 1024

cb171002-d0-Staining 1.003FSC-H ->

0 256 512 768 1024


0 256 512 768 1024


0 256 512 768 1024


0 256 512 768 1024


CD14+

10 10 10 10 100 1 2 3 4

cb171002-d0-Staining 1.003CD3 ->

15,39%

10 10 10 10 100 1 2 3 4

cb171002-d0-Staining 3.005FL1-H ->

58,7%

10 10 10 10 100 1 2 3 4


3,45%

10 10 10 10 100 1 2 3 4

cb171002-d0-Staining 1.00356 ->

3,7%

10 10 10 10 100 1 2 3 4

cb171002-d0-Staining 1.00314 ->

5,71%

T cell B cell NKcells Granulocytes Monocytes

Fluorescence

Side

Sca

tter

Forward Scatter (~ cell size)

Side

Sca

tter

(”gr

anul

arity

“)


CD33+

myeloid lineage

0 256 512 768 1024


10 10 10 10 100 1 2 3 4


6,30%

CD34+

early progenitors

0 256 512 768 1024


10 10 10 10 100 1 2 3 4

cb171002-d0-Staining 3.00534 ->

0,59%

lymphoid lineage

0 256 512 768 1024


CD38+

10 10 10 10 100 1 2 3 4

cb171002-d0-Staining 5.00738 ->

13.73%

Fluorescence

Side

Sca

tter

Forward Scatter

Side

Sca

tter

Flow Cytometry:Progenitors for different lineages in cord blood


apoptotic DNA contentG1 S G2 sub G1 typical for

cells in

From: A Study on triggering apoptosis in T Cells Maria Julieta delPrete1, Thomas Sauer2, Jose Alberto Garcia-Sanz1 and Ernst W. Müllner2

1Department of Immunology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain 2MFPL, Department of Medical Biochemistry, Vienna, Austria

Flow cytometry: DNA profiles

Relative amount of DNA

even

ts


Typical DNA profiles of synchronized cell fractionsobtained e.g. by centrifugal elutriation

G1 G1 G1/S early S mid S late S S/G2 G2 M


fetal liver erythroblasts

3’ LTRMCS

IRES5’ LTR

GFP

hsBcl-XLhsBcl-XL

GFP

79.3%GFP +

Bcl-XLBcl-XL

GFPcontrol

0 48 0 48differentiation (hours)

Bcl-XL overexpression

vector: pMSCV (mouse stem cell virus) gene of interest (anti-apoptotic hsBcl-XL) coupled to GFP via an IRES (internal ribosome entry site); result: rescue of erythroid maturation in the complete absence of differentiation factors

Dolznig, H., Habermann, B., Stangl, K., Deiner, E. M., Moriggl, R., Beug, H., and Mullner, E. W. (2002). Apoptosis protection bythe epo target bcl-x(l) allows factor-independent differentiation of primaryerythroblasts. Curr Biol 12, 1076-1085.

Flow Cytometry: GFP fluorescence in transfected/infected cells Verification of retroviral Bcl-XL gene transfer into primary erythroblasts

Appendix 1: Flow cytometry – detection of apoptosis 1

Differential response of CTLL-2 and B6.1 cells to IL-2 deprivation

Proliferation of both, CTLL-2 and B6.1 cell lines strictly depends on IL-2. Deprivation of this growth factor induces, however, a G1-arrest in B6.1 cells (B) and apoptosis in CTLL-2 cells (A). The DNA content in each cell, at different times after IL-2 withdrawal, was analyzed by FACS, after PI-staining of each cell line, and the fraction of cells in the subG0/G1 phase of the cell cycle was quantified.

(A) CTLL-2 cells deprived of IL-2 show an increase in the fraction of DNA in subG0/G1 already detectable 12 hr after withdrawal, which increases with time.

(B) B6.1 cells do not have a detectable increase in the fraction of DNA in subG0/G1 when deprived of IL-2, even after 62 hr deprivation, although the cells are >95% arrested in G1 (grey). The G1 arrest is reversible, as shown when fresh IL-2 is added to 62 hr deprived cells (black).

From: A Study on triggering apoptosis in T Cells Maria Julieta delPrete1, Thomas Sauer2, Jose Alberto Garcia-Sanz1 and Ernst W. Müllner2

1Department of Immunology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain 2MFPL, Department of Medical Biochemistry, Vienna, Austria


Appendix 2: Detection of apoptosis 2DNA profiles of CTLL-2 and B6.1 cells cultivated +/– IL2 for the times indicated


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