Current Research Applications of Flow Cytometry and Cell

J.Paul RobinsonProfessor of Immunopharmacology Professor of Biomedical EngineeringPurdue UniversityEmail: jpr@flowcyt.cyto.purdue.eduWEB: http://www.cyto.purdue.edu

Faculty Lecture at Kitasato University, Towada, JapanJune 26-July 4, 2000

Lecture summaryThis lecture will discuss the principles of flow cytometry and how they are applied to basic research and clinical questions. We will discuss the general principles of how a flow cytometry operates and why this technology has advantages over many others. In addition, we will look as some examples of newer applications such as apoptosis, multiplexed bead assays and future applications. Cell sorting using your recently acquired Coulter Altra will be described and the key features discussed.

Purdue University Cancer Center&

Purdue University Cytometry Laboratories

What can Flow Cytometry Do?

• Enumerate particles in suspension• Determine “biologicals” from “non-biologicals”• Separate “live” from “dead” particles• Evaluate 105 to 106 particles in less than 1 min• Measure particle-scatter as well as innate fluorescent• Measure 2o fluorescence• Sort single particles for subsequent analysis

Introductory Terms and Concepts Parameter/Variable

• Light Scatter- Forward (FALS), narrow (FS) - Side, Wide, 90 deg, orthogonal

• Fluorescence - Spectral range

• Absorption - loss of light

• Time - Kinetics

• Count - number of events/particles/cells

Concepts

Scatter: Size, shape, granularity, polarized scatter (birefringence)

Fluorescence: Intrinsic: Endogenous pyridines and

flavinsExtrinsic: All other fluorescence

profiles

Absorption: Loss of light (blocked)Time: Useful for kinetics, QCCount: # events -always part of any collection

Instrument Components

Electronics: Control, pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control

Optics: Light source(s), detectors, spectral separation

Fluidics: Specimen, sorting, rate of data collection

Data Analysis: Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation

Arc Lamp Excitation SpectraIr

rad

ian

ce a

t 0.

5 m

(m

W m

-2 n

m-1)

Xe Lamp

Hg Lamp

Shapiro p 99Shapiro p 99

Lasers• Argon laser

• He-Ne Laser

Optical Collection systems

He-Cd Laser Argon Laser He-Ne Laser2nd Argon Laser

Elite Cytometer with 4 Lasers

Water cooled argon laser

He-Cd laser

Air-cooled argon laser

Santa clause

Optical DesignOptical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

Coulter Optical System – Elite/AltraCoulter Optical System – Elite/Altra

• The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted

555 - 595

PMT4

APC 655 - 695

PMT6

PMT7

49

0

DL

488

BK

05

5

DL

62

5

DL

675

BP

488 BP525 BP575 BP

Purdue Cytometry Labs PUCL3034

632

BP

TM

PMT3 PMT2 PMT1

PMT5

Fluidics

SMALL BEAD LARGE BEAD

Frequency Histogram

SMALL BEAD LARGE BEAD

Sample inSheath

Sheath in

Laser beam

Stream Charge

+4KV -4KV

Waste

SORT RIGHTSORT LEFT

SORT DECISIONS

Piezoelectriccrystal oscillator

Last attacheddroplet

LEFT RIGHT

Sensors

Sensor

Signals are collected from several sensors placed forward or at 90° to the laser beam. It is possible to “sort” individual particles. The flow cell is resonated at a frequency of approximately 32KHZ by the piezoelectric crystal mounted on the flow cell. This causes the flowing stream to break up into individual droplets. Gating characteristics can be determined from histograms (shown right) and these can be used to define the sort criteria. These decisions are all controlled by the computer system and can be made at rates of several thousand per second.

Figure 1 The central component of a flow cytometer is the flow cell. A cutdown of a typical flow cell indicates the salient features. Sample is introduced via the sample insertion rod. Sheath fluid (usually water or saline) is ntroduced to surround the insertion rod causing hydrodynamic focussing of flowing cells which are contained within a core fluid. The laser intersects the fluid either outside the flowcell (in air) or in a slightly extruded portion of the flow cell tip (in quartz).

Fluorescence• The wavelength of absorption is related to

the size of the chromophores

• Smaller chromophores, higher energy (shorter wavelength)

Fluorescence• Stokes Shift

– is the energy difference between the lowest energy peak of absorbance and the highest energy of emission

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Flu

ores

cnec

e In

tens

ity

Wavelength

Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines

Fluorescence

Resonance Energy Transfer

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

Flow cytometry measurements

L

M

G

SCATTER FLUORESCENCE IMAGE

Data Presentation Formats

• Histogram• Dot plot• Contour plot• 3D plots• Dot plot with projection• Overviews (multiple histograms)

Data Analysis Concepts

Gating • Single parameter• Dual parameter• Multiple parameter• Back Gating

Note: these terms are introduced here, but will be discussed in more detail in later lectures

FITC Fluorescence

Mo1

CD4 CD8

CD8

CD45

leu11a

CD20 Tube

ID

The Cell Cycle

G1

MG2

S G0Quiescent cells

A DNA histogram

G0-G1

S

G2-M

Fluorescence Intensity

Cel

l Num

ber

A typical DNA Histogram

G0-G1

S

G2-M

Fluorescence Intensity

# of

Eve

nts

log Thiazole Orange.1 1000 100 10 1

Count

0

150

112

75

37

RMI = 0RMI = 0

log Thiazole Orange.1 1000 100 10 1

Count

0

150

112

75

37

RMI = 34RMI = 34

Reticulocyte Analysis

Labeling Strand Breaks with dUTP [Fluorescein-deoxyuridine triphosphate (dUTP)]

Green Fluorescence is Tdt and biotin-dUTP followed by fluorescein-streptavidinRed fluorescence is DNA counter-stained with 20µg/ml PI

PI-Red Fluorescence

Green Fluorescence

Green Fluorescence

Sid

e S

catt

er

Forward Scatter

Green:apoptotic cells

Red:normal cells

R2: Apoptotic Cells

R1: Normal Cells

Scatter Pattern of Human leukocytes

Lymphocytes

Monocytes

NeutrophilsA flow cytometryscattergram

For

war

d sc

att e

r (s

ize)

Side scatter (granularity)

Three Color Lymphocyte Patterns

CD3

CD

4

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD4 -->

CD3

CD

4

CD8C

D8

10 1 10 2 10 3 10 4

CD8 -->

101

102

103

104

CD4 -->

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD8 -->

Data from Dr. Carleton Stewart

YoYo-1 stained mixture of 70% ethanol fixed E.coli cells and B.subtilis (BG) spores.

mixture

BG E.coli

BG

E.coli

mixture Run on Coulter

XL cytometerS

catt

er

Fluorescence

Sca

tter

Live cell/dead cell PI Fluorescence

Data from Dr. Doug Redelman, Sierra Cytometry

PIHoechst 33342

Oxidative Reactions

• Superoxide Hydroethidine• Hydrogen Peroxide

Dichlorofluorescein• Glutathione levels Monobromobimane• Nitric Oxide Dichlorofluorescein

Calcium Flux

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200

Rat

io: in

tens

ity o

f 46

0nm

/ 4

05nm

sig

nals

Time (seconds)

Time (Seconds)0 36 72 108 144 180

RATIO [short/long]

0

200

400

600

800

StimulationStimulation

Flow Cytometry Image Cytometry

Membrane Potential• Oxonol Probes • Cyanine Probes

How the assay works:• Carbocyanine dyes released into the surrounding media as cells depolarize

• Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs

Time (sec)

Gre

en F

luor

esce

nce

Repolarized Cells

051

210

24

0 300 1500 1200 2400Time (sec)

051

210

24G

reen

Flu

ores

cenc

e

PMA Added fMLP Added

Depolarized Cells

Summary

Main Applications

• DNA and RNA analysis

• Phenotyping

• Cell Function

• Sorting and cell isolation

• Immunological assays

The facilities at Kitasato University

Coulter AltraThe facilities at Kitasato University

Coulter XL CytometerThe facilities at Kitasato University