FACS Analyse von Zytokinen

Oliver Pelz-AckermannApplication Specialist BD Biosciences

Zytokine in Lösung - BD™CBA

(=Cytometric Bead Array)

1. CBA Definition

•

Fluorescent immuno-assay allowing the simultaneous detection and quantification of proteins within one sample using flow cytometry.

•

The system is composed off various beads with different fluorescence intensity coated with high affinity capture antibodies.

•

The system allows multiplex analysis.

•

The data acquired on a flow cytometer is analysed using a specific software.

How a bead based assay works

+

+

Analyte of Interest Fluorescent Detector Ab

Capture BeadCapture Ab

Multiplexing with beads: The Theory

•

How?

Size

Color

Intensity

The BD Way of Multiplexing

•

One size: 7.5um

•

One or two colours: Kits vs. Plexes

•

Different intensities:

6 for the BDTM

CBA kits

30 for the BDTM

CBA Flex Set Plexes

Key Features to Multiplex using Beads

•

Each antibody pair needs to be very target specific. No cross-reactivity or cross-inhibition.

•

An ELISA antibody pair may not work on beads. Coupling chemistry can influence the binding capacity of an antibody.

•

An antibody pair should give a minimal fluorescence intensity and a maximal sensitivity and reproducibility.

•

Detection antibodies will preferably be directly conjugated to a stable and bright fluorochrome: PE.

Two steps detection systems using biotin-streptavidin-PE can increase background and decrease the sensitivity of the assay

Principle of BDTM

CBA Kits

•

CBA quantitates

specific solutes in sera, plasma, tears or tissue culture supernatants

•

Based on sandwich ELISA, with a capture and detection Ab.

•

However, with CBA 50ul of sample is enough to measure 6 different cytokines. Conventional ELISA uses 100ul sample/cytokine.

•

The data is acquired in a flow cytometer and analysed

using the FCAP Array Software.

•

Capture Ab

bound to Fluorescent beads

6 beads that emit light on FL-3. All same size but different fluorescent intensities.

Each bead is bound by capture antibodies like an ELISA well in a 96-well plate.

300 events are acquired/capture bead.

•

Detection antibody coupled to PE.

Detection antibody emits light on FL-2.

The more soluble analyte present, the more detection antibody binds, and the brighter the PE signal

FL3

FL2

FL2

Principle of BDTM

CBA Kits

One Step

3 h incubation

Principle of BDTM

CBA Kits

Sample Acquisition

BD FACSCaliburTM

BD FACSArray TM

Sample Analysis with FCAP Software

Principle of BDTM

CBA Kits

Principle of BDTM

CBA Flex Sets

•

Capture Ab

bound to Fluorescent beads

30 beads that emit light on FL-3 and FL-4. All same size but different fluorescent intensity mixtures.

Each bead is bound by capture antibodies like an ELISA well in a 96-well plate.

300 events are acquired/capture bead.

•

Detection antibody coupled to PE.

Detection antibody emits light on FL-2.

The more soluble analyte present, the more detection antibody binds, and the brighter the PE signal FL2

FL3

FL3

FL4

4 5 6 7 8 9A

B

C

D

E

Principle of BDTM

CBA Flex Sets

•

Soluble and Cell signaling human, mouse and rat BD™

CBA Flex Set

Assays:

Antibody-conjugated capture bead

PE-detection reagent

2 standards

•

BD™

CBA Flex Set Master Buffer Kit

Assay reagents

Instrument setup beads

•

FCAP Array™

multiplex analysis software

Principle of BDTM

CBA Flex Sets

InstrumentInstrument LasersLasersBead Bead

EmissionEmission DetectorDetectorData Data

processingprocessingNumber Number

of plexesof plexes

CaliburCalibur 488nm635nm

FL-3FL-4

PEAnalog 30

ArrayArray 532nm635nm Red and NIR

YellowDigital 30*

Canto/LSRCanto/LSR

Fortessa/AriaFortessa/Aria

488nm633nm APC and

APC-Cy7

PEDigital 30*

Principle of BDTM

CBA Flex Sets

Total 3 hours

1 hour 2 hours

Soluble CBA Flex

Cell Signaling CBA Flex (cell lysate)

Total 4 hours

3 hours 1 hourBeads

in bead diluentw/o colors

Beads +

Sample/standardwith assay diluent

yellow

Beads + Sample/standard +

detectorwith detector diluent

= green

Principle of BDTM

CBA Flex Sets

Sample Acquisition

FACS Instrument

BD FACSArrayTM

Sample Analysis with FCAP Software

Data Acquisition on different instruments Instrument Set-up Manuals for BDTM

CBA Flexes

BD FACSCaliburTM

http://www.bdbiosciences.com/pdfs/manuals/23-8653-00-.pdf

BD FACSArrayTM

http://www.bdbiosciences.com/pdfs/manuals/23-8654-00-.pdf

BD FACSDiVaTM

based instruments (BD FACSCanto™, BD™

LSR II, and BD FACSAria™)

http://www.bdbiosciences.com/docs/23-8795-00.pdf

Summary -

CBA vs. CBA Flex

CBA CBA Flex

No. of beads 6 30*

Bead fluorescence

FL-3 (670nm)

FL-3 (670 or 785) FL-4 (660)

Detection method PE PE

Sample In solution In solution Cell lysate (CBA Cell Signaling)

Flow cytometer 1 laser 488nm

2 lasers : 488 & 635 or 532 & 635

Software FCAP v3.0 FCAP v3.0Red/FL-4

NIR

/ FL-

3

A

B

C

D

E

4 5 6 7 8 9

Experimental Data

Experimental Data 30 plexes

with human PBMCs

PBMCs

• plate-bound anti-hCD3 (#555329)• soluble anti-hCD28 (#555725)• rhIL-2 (#554603)• rhIL-4 (#554605)

2 days Wash

PMA + Ionomycinor

anti-hCD3 and anti-hCD2824 hrs

rhIL-2 (#554603)rhIL-4 (#554605)

3 days Wash

•

Collection of supernatants•

CBA Flex Analysis of IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-

12p70, IL-13, IFNg, TNF, LT-α, G-CSF, GM-CSF, RANTES, VEGF, IP-10, MIP- 1α, MIP-1β,

MCP-1, MIG, Angiogenin, bFGF, FasL, Eotaxin, OSM and

IgE.

Calibur: Save FCS File from File Menu

FACSArray:

Experimental Data 30 plexes

with human PBMCs

PBMCs in blood

30 ELISA Samples

Experimental Data 30 plexes

with human PBMCs

IL-2

IL-4

IL-5

IL-6

IL-7

IL-8

IL-1β

IL-10

IP-10

IL-9

VEGF

MIP-1α

Angiogenin

bFGF

FasL

Eotaxin

G-CSF

GM-CSF

RANTES

IL-3OSM

LT-αMCP-1

TNF

MIP-1β IL-12p70

IL-13IFN-γMIG

IgE

1 CBA Sample

PBMCs in blood

Experimental Data 30 plexes

with human PBMCs

Experimental Data 30 plexes

with human PBMCs

Experimental Data CBA Flex vs. ELISA

•

Interferences has been described

due to

Cross-reactivitiy

with other cytokines [Keller et

al, JIM 2003, 279; 277-285]

Cross-reactivity to cross-species antibodies [Keller et al, JIM 2003, 279; 277-285 and Earley

et al, Cytometry 2002; 50:239-242]

Interference with other “substances”

[Pang et al, JIM 2005; 302:1-12]

Cross-reactivity and sensitivity issues ?

Major Differences ELISA CBA

Reporter SystemEnzyme Amplification of a

colorimetric substrate Fluorescence

Capturing Flat surface of a 96-well plate Spherical beads in suspensionTechnology Single Plex Multiplex

Sample specificSample specific

Experimental Data CBA Flex vs. ELISA

•

Comparison of ELISA vs. CBA using the same capture and reporting antibodies

Chen R, Lowe L, Wilson JD, Crowther E, Tzeggai K, Bishop JE, Varro R. Simultaneous Quantification fo Six Human Cytokines in a Single Sample Using Microparticle-based Flow Cytometric Technology. Clin Chem 1999: 45 (9): 1693-1694

Experimental Data CBA Flex vs. ELISA

•

Comparison of ELISA vs. CBA using different capture and reporting antibodies (ELISA R&D, CBA (BD Biosciences)

Tarnok

A, Hambsch

J, Chen R, Varro R.Cytometric Bead Array to Measure Six Cytokines in Twenty-Five Microliters

of Serum, Clin

Chem

2003 49(6): 1000-1001

Experimental Data Different techniques

Method Western Blot ELISAEliSpot Assay

Immuno-precipitation

Immuno-cytochemistry

Immuno-histochemistry

Flow- cytometry

Mulitplex Assay

Target (Analyte)Proteins or

polypeptidesPeptides, proteins,

others CellsProteins or complexes Cells

Tissue sections or tissue array Cells

Proteins, other

Data FormsProtein banding

image Numeric and curveImage or numerical

Protein binding image Image Image

Numerical or curve

Numerical or curve

Sensitivity ng/ml pg/ml pg/ml ng/ml ng/ml ng/ml 1/10E5 cells pg/ml

Reproducibility Good High Good Variable Variable Variable Good HighQuantification Semi Yes Semi Semi Semi Semi Yes Yes

High Troughput No Possible No No No Possible Yes Yes

Summary

Summary of CBA technology

-

Samples used with Soluble CBA/CBA Flex

Pleural fluid

•

Hiraki

AK, Murakami T, Murakami K, Makihata

K, Takao K, Eda

R, Maeda T, Sugi

K, Darzynkiewicz

Z, Takeyama H. Relative abundance and patterns of correlation among six cytokines in pleural fluid measured by cytometric bead array, Int

J Mol Med 2003 (12/2):193-8.

Vitreous fluid•

Maier R, Weger

M, Haller-Schober

EM, El-Shabrawi

Y, Theisl

A, Barth A, Aigner

R, Haas A. Application of multiplex cytometric bead array technology for the measurement of angiogenic factors in the vitreous, Mol Vis. 2006 (2/12):1143-7.

Breath condensate•

Gessner

C,Rechner

B,Koker

J,Kuhn

H,Hammerschmidt

S,Sack

U,Wirtz

H. Erhöhte

Konzentrationen

von VEGF, bFGF

und Angiogenin

im

Atemkondensat

bei

Patienten

mit

einem

nicht-kleinzelligen

Lungenkarzinom

(NSCLC), Pneumologie

2007: 61

Summary of CBA technology

-

Samples used with Soluble CBA/CBA Flex

CFS (Cerebrospinal fluid)

•

Fragoso-Loyo

H,Richaud-Patin

Y, Orozco-Narváez

A, Dávila-Maldonado L, Atisha-Fregoso

Y, Llorente

L, Sánchez-Guerrero J. Interleukin-6 and chemokines in the neuropsychiatric manifestations of systemic lupus erythematosus, Arthritis & Rheumatism, 2007 (56/4): 1242-

1250.

Urine•

Jiménez

R, Ramírez

R, Carracedo

J, Agüera

M, Navarro D, Santamaría

R, Pérez

R, Del Castillo D, Aljama

P. Cytometric bead array (CBA) for the measurement of cytokines

in urine and plasma of patients undergoing renal rejection, Cytokine, 2005 (7/32(1)):45-50.

Quantification of viral proteins•

Yan X, Schielke

EG, Grace KM, HassellC, Marrone

BL, Nolan JP. JIM, 2004 (284):27-38,

Bronchoalveolar

lavage•

Ema

A, Emad

Y. Levels of Cytokine in Bronchoalveolar

Lavalge

(BAL) Fluid in Patients with Pulmonary Fibrosis Due to Sulfur Mustard Gas Inhalation, J Interferon & Cytokine Research, 2007 (1): 38-43.

Example –

LSR Fortessa

Example –

LSR Fortessa

Example –

LSR Fortessa –

FCAP v3.0

Example –

LSR Fortessa –

FCAP v3.0

Example –

LSR Fortessa –

FCAP v3.0

Zytokine in der Zelle - Intrazelluläre Zytokinfärbungen

Intracellular Cytokine Staining

Advantages of intracellular cytokine analysis:

Analysis is less time consuming compared to ELISA or ELISPOT

Multicolor

cytometry

with co-staining of surface markers enables cytokine profiling of subpopulations

Cytokine analysis can be multiplexed (eg. Quantification of Th1, Th2 and Th17 cells in in one sample)

Quantification of cells that contribute to cytokine production

Requirements for successful Cytokine Analysis

•

Use cells that are able to produce the desired cytokine and choice of suitable activation method

•

Addition of an inhibitor of secretion for intracellular accumulation of the cytokines

•

Appropriate fixation and permeabilisation protocol

•

Run

appropiate controls

•

Use of bright fluorochromes to detect cytokines

Cell activation

•

Unspecific Activation

easier to perform

significantly stronger

PMA/Ionomycin, PHA/ConA, LPS

•

Specific Activation

Simulation of the in vivo situation. Relevance of results is higher

Antigen, MLR (MHC contact), TCR (CD3±CD28), Cytokines (IL-2, IFN)

•

Information on cell types and activation protocols:

Application Handbook (chapter 4)

•

http://www.bdbiosciences.com/pdfs/manuals/02-8100055-21B.pdf

Website (technical data sheets)

•

http://www.bdbiosciences.com/pharmingen/protocols/Activation.shtml

Cell activation

2 hrs IFN-+ LPS / Golgi Plug

16 hrs

Human MonocytesIL

-12

(PE)

Autofluorescence (FL1)

LPS +Golgi Plug

4 hrs

LPS +Golgi Plug

16 hrs

•

Activated and fixed Mouse, Human and Rat cell populations

Cell activation: Positive controls

Cytokine secretion retardation

•

Cytokines are rapidly secreted upon expression which results in

low / not detectable intracellular concentrations.

Use of Secretion Inhibitors for Accumulation:

GolgiPlug

# 555029 (Brefeldin

A)

GolgiStop

# 554724 (Monensin)

•

Secretion inhibitors are toxic. Exposition of cells should be as short as possible! (2-24h)

GolgiStop: -

+

Cytokine secretion retardationB

D G

olgi

Plug

Bre

feld

inA

BD

Gol

giSt

opM

onen

sin

Superior Superior

Superior

Equal

Comparable Cytokine-Detection for: IL-2; IL-3; IL-5; IL-6; IL-12; GM-CSF

Cell Fixation and Permeabilization

•

Fixation 3-4% Paraformaldehyde

(PFA)

•

Permeabilization

0.05 –

0.2% Saponin

(in some cases other detergents might be better)

Saponin

permeabilization

is reversible

Needed for all steps•

Washing steps

•

Antibody incubation

Blocking Unspecific Antibody Binding

•

Reagents that block Immunoglobulin (Ig) Fc

receptors:

In the mouse and rat systems: purified 2.4G2 and D34–485 antibodies directed against FcγII/III (mouse BD FcBlock™; #553142 and #553141) and Fcγ

receptors (rat BD FcBlock

CD32

#550271 and #550270)•

Preincubate

cell suspension with 1 μg

BD FcBlock/106

cells in 100 μl of staining buffer for 15 min at 4°C.

In human cells

: excess of irrelevant, purified polyclonal Ig

or 10% normal human serum or polyclonal human IgG

(Sigma Cat. No. I-

4506 -

Preincubate

1-10ug/106

cells in PBS for 20 minutes at 4°C to block Ig

Fc

receptors)

Negative Controls

•

Unstimulated

cells

•

Isotype

controls and/or “fluorescence minus one” controls (FMO)

•

Additional controls:

Ligand

Blocking Control:

Pre-block anti-cytokine antibody with excess of recombinant cytokine protein (eg. 0.25µg per test)

Unconjugated

Antibody Control:Pre-incubate cells with an excess of un-conjugated antibody from the same clone (eg. 5µg per test)

Fluorochromes

for Intra-

and Extra-

Cellular Staining

Use bright fluorochromes

for staining of cytokines or activation markers

Cytokine: PE, APC or Alexa

Fluor 647, PE-Cy7

Activation marker: APC, PE or Alexa

Fluor 647•

Retardation might affect transport to surface

Lineage marker: FITC, PerCP, PerCP-Cy5.5, APC-H7, Pacific Blue or any other suitable fluorochrome

•

Tandem conjugates such as PE-Cy5, PE-Cy7 or APC-Cy7 might not be compatible with fixation

Intracellular Cytokine Staining: Summary

Find appropriate stimulus for cells of interest•

optimize concentrations and activation time •

Use positive controls (unspecific stimulation or prestimulated

control cells)

Retardation of cytokines•

choose between Monensin

and Brefeldin

A

Fixation and permeabilization

of cells (one protocol for all)

Blocking of unspecific antibody binding

Find appropriate antibodies and fluorochrome

combinations for•

cytokine of interest and activation marker (bright fluorochromes)•

lineage marker (any other suitable fluorochrome)

Chose appropriate negative control•

Unstimulated

cells•

isotype

controls and/or “FMO”

for cytokine-antibodies

Suitable gating strategy for analysis of subpopulations

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