Rui Gardner ruig@igc.gulbenkian.pt

Applications in Flow Cytometry

Cláudia BispoIGC – April 4, 2013

Outline

• Immunophenotyping

• Cell cycle

• Apoptosis

• Cell proliferation

• Cell activation

• Calcium flux

• Cytokine Secretion

• Activation of signalling pathways

• Levels of intracellular reactive oxygen species

Cell State

Cell Function

• Dead/Live Discrimination

• Absolute counting

• Time points

Microbiology

Potential Applications of Flow Cytometry

Evaluate Cell State

Immunophenotyping

• Uses labeled antibodies (Abs) to identify cells of interest

• Determination of cell surface antigens• Allows for detailed identification of cellular subsets

(simultaneously measure multiple parameters cell by cell)

• Targets on both surface and intracellularly

CAUTION – Abs selection:Fluorophore’s excitation

spectrum must match the laser line used, and its emission must fall within detection filter sets in

the cytometer

Cell Cycle

DNA content analysis - Propidium Iodide (PI)

G2

M

G1

S

G0

G0/G1

S-phase

G2/M

Fluorescence (DNA content)

Excitation / Emission : 488nm / max 617nm

Cell Cycle Analysis

Cell Cycle Analysis Software

G0/G1

SG2/M

Fluorescence Intensity

Cell

Num

ber

• Accurate measurements allow for resolution of normal cells undergoing G1, S, G2 phases• Also useful when multiple DNA populations present: measuring aneuploidy & polyploidy

Examples:FlowJoModFit LT FCS Express IDLYK …

Cell Cycle - Bromodeoxyuridine (BrdU) method

Propidium Iodide plus BrdU staining

• BrdU is thymidine analog

• Taken up by cells in S-phase

• Usually in combination with PI

0 200 400 600 800 1000FL3-H

Anti -

Brd U

FI T

C

G1 G2/M

S Phase

101

102

103

104

Propidium Iodide

Excitation / Emission : PI 488nm / max 617nmBrdU varies by fluorophore

Cell Cycle - G0/G1 discrimination

Pyronin Y plus Hoechst 33342/33258

G0/G1

S

G2/M

G0

S

G2/M

G1

Cell

Coun

tRN

A Co

nten

t

Excitation / Emission : PY 488nm / 575nmHO UV line / 460-490nm

DNA content (A-T base pairs)

RNA content

Apoptosis

Morphological Changes Functional/Biochemical

Cell shrinkage Cell shape change Condensation of cytoplasm Nuclear envelope changes Nuclear fragmentation Loss of cell surface structures Apoptotic bodies Cell detachment Phagocytosis of remains

Free calcium ion rise bcl2/BAX interaction Cell dehydration Loss of mitochondrial membrane potential Proteolysis Phosphatidylserine externalisation Lamin B proteolysis DNA denaturation 50-300kb cleavage Intranucleosomal cleavage Protein cross-linking

Changes in light scatter

DNA denaturationChanges in plasma

membraneChanges in cell organelles /

signaling pathways

* positive control is useful

Apoptosis

CELL DEATH – FSC x SSC

0 200 400 600 800 1000FSC-H: FSC-Height

100

101

102

103

104

SSC

-H: S

SC-H

eigh

t (Lo

g Sc

ale)

27.6

0 200 400 600 800 1000FSC-H: FSC-Height

100

101

102

103

104

SSC

-H: S

SC-H

eigh

t (Lo

g Sc

ale)

37.4

0 200 400 600 800 1000FSC-H: FSC-Height

100

101

102

103

104

SSC

-H: S

SC-H

eigh

t (Lo

g Sc

ale)

15.9

0 200 400 600 800 1000FSC-H: FSC-Height

100

101

102

103

104

SSC

-H: S

SC-H

eigh

t (Lo

g Sc

ale)

17.1

Via

bilit

yA

ctiv

atio

n

Medium 100nM Rapa

27.6% 15.9%

37.4% 17.1%

0 200 400 600 800 1000FSC-H: FSC-Height

100

101

102

103

104

SSC

-H: S

SC-H

eigh

t (Lo

g Sc

ale)

26.2

0 200 400 600 800 1000FSC-H: FSC-Height

100

101

102

103

104

SSC

-H: S

SC-H

eigh

t (Lo

g Sc

ale)

26.1

0 200 400 600 800 1000FSC-H: FSC-Height

10 0

10 1

10 2

10 3

10 4

SSC

-H: S

SC-H

eigh

t (Lo

g Sc

ale)

28.8

0 200 400 600 800 1000FSC-H: FSC-Height

100

101

102

103

104

SSC

-H: S

SC

-Hei

ght (

Log

Scal

e)

25.6

Via

bilit

yA

ctiv

atio

n

Medium 100nM Rapa

26.2% 25.6%

26.1% 28.8%

T-ALL Thymocytes PBMCs

Changes in light scatter: low level resolution of apoptotic cells

Apoptosis

Propidium Iodide (fixed cells)

DNA degradationDNA Degradation

Other viability dyes :• 7-AAD• Zombie Aqua• To Pro3‐• ...

Apoptosis

Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)

Apoptosis

Annexin V plus Propidium Iodide

Excitation / Emission : Annexin varies by fluorophorePI 488nm / 617nm

Apoptosis (intracellular staining)

Fix and permeabilize Add

Antibody

Analysis by Flow

Cytometry

Apoptosis – Bcl-2 family members

Excitation / Emission: Varies by fluorophore

Apoptosis – Activated forms of Caspases

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate).

UntreatedEtoposide

Excitation / Emission: Varies by fluorophoreEx A488: 488nm / 520nm

Tracking Cell Proliferation with CFSE (Carboxyfluorescein Succinimidyl ester)

STAIN WITH CFSE

Dilution of CFSE

Cell Divisions

CELL

Cell Proliferation

Excitation / Emission: 488nm / 521 nm

1234 0

Tracking Cell Proliferation with CFSE

IL-7 IL-7+ DMSO

IL-7+ PI3K Inhibitor IL-7+ Erk Inhibitor

Evaluate Cell Function

Cell Activation

FSC x SSC – Cell size

Sofia Marques (IMM)Filipa Lopes (IMM)

Cell Activation

Activation markers: CD69, CD71, etc

Excitation / Emission: Varies by fluorophore

Calcium Flux

Fluorescence-imaging of human erythrocytes treated with PGE2 using the calcium fluorophor Fluo-4

Effects of T cell receptor stimulation by B-Cells on ionized calcium concentration ([Ca2+]i).

Sofia Marques (IMM)Filipa Lopes (IMM)

Excitation / Emission:Indo1 UV line / 405nmFluo-4 488nm / 516nm

Activation of signalling pathways

Krutzik et al. Clin Immunology (2004)

Activation of signalling pathways

Phospho-protein detection

• Ability to analyze rare subsets of cells within complex populations

• Better evaluate population heterogeneity

Krutzik et al. Clin Immunology (2004)

• Discrimination of High vs. Low responders

pStat1

Combining Surface Markers with Phospho-staining

Evaluate protein phosphorylation in different subpopulations concurrently

Levels of intracellular reactive oxygen species

Measurement of oxidative burst in human peripheral blood granulocytes using dihydroethidium. On oxidation, ethidium is produced which binds to DNA & fluoresces red.

The histogram shows the red fluorescence before and after incubation with 10 mM muramyl dipeptide for 15 min.

Excitation / Emission:488nm / 605nm

Cytokine Secretion - Multiplex Bead Arrays

Luminex MAGPix System

Quantitation & detection of cytokine and signal transduction proteins, DNA & RNA

- Magnetic bead-based technology

- Up to 50 analytes /well of a 96 well plate

- Up to 4.800 results in 1 hour

- Uses small sample volumes

SOON@ UIC

bead coated with capture antibody mixture

Multiplex Bead ArraysSOON@ UIC

Or design custom and personalized panels

Merck Millipore

MAGPix

Microbiology Applications

Microbiology - Dead/Live Staining

• Various established methologies can be optimizedo SYTO 9 /PI - Live/Dead BacLight viability kito SYBR Green-I /PI - Barbesti (2000)o DiBAC4(3)/EB/PI - Flow Cytometry Protocols 2nd ed.o etc...

• Several kits available

CAUTION:Make sure excitation/emission of kit dyes do not coincide with the

ones on your sample

LIVE/DEAD kit for Bacteria LIVE/DEAD kit

for Yeast & Fungi

Excitation / Emission: 488nm / Varies by method

Microbiology

Monitoring Cell Cycle – DNA contentMinor protocol adjustments/optimization might be needed comparatively with established protocols for eukaryotes

E.coli: DRAQ5 - Silva et al. (2010) Yeast : PI or SYTOX Green - Knutsen (2011)

Excitation / Emission: 647nm / 670nm

Excitation / Emission:488nm / 520nm

Time PointsAssess treatment effect on population survival, protein expression, etc at indicated time points

- effect on protein expression

- counting: population proportions

Microbiology

CAUTION:Make sure you use a

fluorescence control when protocol requires measuring & comparing protein expression

Cell counting

• Employs the use of reference beads on a regular cytometer• Allows the examination of large number of cells per sample• Combining Surface Markers will allow to count specific subpopulations

• Handheld Automated Cell Counter• Allows cell differentiation by its volume (size) according to

the Coulter principle• Uses a disposable sensor

Scepter

Beads

@ UIC

Cell counting (2)

• Countess™ Automated Cell Counter (Invitrogen)• Tali™ Image-Based Cytometer (Invitrogen)• TC20 Automated Cell Counter (BioRad)• etc...

Image based alternatives

@ UICSOON@ UIC

Today’s Future Applications

Amnis Image Stream Merck Millipore

Amnis Image Stream

CyTOF – Mass Cytometer

Omatsky et al. JAAS (2008), Bendall et al. (2011)

• Can measure 34 parameters simultaneously in a single cell• Cells are labeled with Abs containing rare elements metal tags• Cells are atomized & ionized in plasma (high temperatures)• Tags are separated & identified by mass (time-of-flight)

Future Advances

• Heading further into the path of Single Cell Analysis– microfluidics & lab-on-a-chip systems

• Reduced laser size and capillary flow techniques mean smaller instruments

• Instruments can now image cell at point of laser interrogation

• More colours for immunofluorescence

Summary Flow Lab Equipment @ UIC

Available Laser Lines at IGC’s Flow Cytometry Lab

Bench Top Analyzers Cell Sorters

FACScan FACScalibur CyAn ADP

LSR Fortessa MoFlo FACSAria

Multiline UV (330-360 nm) Violet (407 nm)

Blue Violet (442 nm) Cyan (457 nm) Blue (488 nm)

Green (514 nm) Yellow(561 nm) Red (640 nm)

Summary of applications @ UIC Flow Lab

CytometerImmuno-

phenotyping / activation

state

Cell Cycle / Cell

proliferationApoptosis Calcium flux

(with Fluo-4)Multiplex cytokine secretion

Activation Signaling

Pathways / ROS

Microbiology applications

FACSCalibur

FACSScan

CyAn ADP

BD Fortessa Luminex MAGPix