Flow Cytometry 101 - Purdue University Cytometry Laboratories

breadloafvariousΒιοτεχνολογία

20 Φεβ 2013 (πριν από 4 χρόνια και 6 μήνες)

146 εμφανίσεις

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Immunophenotyping and

applications of cell analysis in the
hematology laboratory

J.Paul Robinson

Professor of Immunopharmacology &
Biomedical Engineering

April 5, 2005

This lecture can be found on

http://www.cyto.purdue.edu/class

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

What can flow cytometry be used for?


Immunology


Hematology


Pathology


Microbiology


Genetics


Drug discovery


Toxicity testing


Cell culture studies


Functional studies


Clinical and Research


Chemical Engineering


Biotechnology


Agronomy


Animal Sciences

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

CELLULAR ANTIGENS

Adhesion

Receptors

Metabolic

cytokines

structure

enzymes

Slide courtesy of Jim Bender

T cells

B Cells

Phenotype: …outward physical manifestation…

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Cluster Designations (CD)


These are based on the Immunology Workshop an
international committee that meets in Boston
every few years


Each antigen that is defined on cells is given a
unique number


Until a final number is agreed, antigens can be
designated CDw (w=workshop a tentative
designation)


Here is an example of the
possible CDs

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Immunofluorescence staining

specific binding

nonspecific binding

Slide from Dr. Carleton Stewart

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Direct staining


Fluorescent probe
attached to antibody


Specific signal:
weak, 3dyes/site


Nonspecific binding:
low

Slide from Dr. Carleton Stewart

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Avidin
-
Biotin method I

biotinylated
primary Ab

biotin

avidin

biotinylated dye

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

CFU
-
GM

MYELOBLAST

MYELOCYTE

META
-

MYELOCYTE

BAND

PMN

CD16

CD11b

CDw13

CD33


CD34

HLA
-
Dr


CD38

CD71

MY8

Myelomonocytic Antigen Distribution

Purdue Cytometry Labs

PROGRANULOCYTE

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Negative

Positive

Decision Tree in Acute Leukemia

HLA
-
DR

T

CD13,33

CD19

TdT

CD10

CD20

Mu

B,T

AMLL

AML

T
-
ALL

AML
-
M3

AUL

?

PRE
-
BI

PRE
-
BII

PRE
-
BIII

PRE
-
BIV

PRE
-
BV

CD13,33

From Duque et al, Clin.Immunol.News.

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

What are the principles in flow
cytometry?


Light

scatter
ed by a laser or arc lamp


Specific
fluorescence

detection


Hydrodynamically focused stream

of particles


Electrostatic particle separation

for sorting


Multivariate data analysis

capability

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Concepts

Scatter
:

Size, shape, granularity, polarized


scatter (birefringence), structure


Fluorescence
:


Intrinsic
: Endogenous pyridines and flavins


Extrinsic
: All other fluorescence profiles


Absorption
: Loss of light (blocked)

Time
:


Useful for kinetics, QC

Count
:

Number of cells collected in a


histogram


Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Clinical Analyzers

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Cell Sorters (FACS


Fluorescence Activated Cell Sorter)

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor





Optical Design

PMT 1

PMT 2

PMT 5

PMT 4

Dichroic

Filters

Bandpass

Filters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Hydrodynamic Systems

Sample in

Sheath

Sheath in

Laser beam

Piezoelectric

crystal oscillator

Fluorescence

Sensors

Scatter Sensor

Core

Sheath

Signal

direction

Flow Chamber

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Light Scatter


Materials scatter light at wavelengths at which they do
not absorb


If we consider the visible spectrum to be 350
-
850 nm
then small particles (<
1/10


) scatter rather than absorb
light


For small particles (molecular up to sub micron) the
Rayleigh scatter

intensity at 0
o

and 180
o

are about the
same


For larger particles (i.e. size from 1/4 to tens of
wavelengths) larger amounts of scatter occur in the
forward not the side scatter direction
-

this is called
Mie
Scatter

(after Gustav Mie)
-

thus forward scatter is
related to size (at 1
-
15 microns)


Shapiro p 79

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Optics for forward scatter


scatter

detector

iris

blocker

Laser
beam

Stream in air or a
round capillary

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Frequency distribution

Number of events

Intensity of parameter (e.g. fluorescence)

histogram

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

histogram

Intensity of parameter

Number of events

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Flow cytometry measurements

L

M

G

SCATTER







FLUORESCENCE




IMAGE

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

0


200


400


600


800

1000

0
200
400
600
800
1000


Side Scatter Projection

Forward Scatter Projection
Light Scatter Gating

Forward Scatter Projection

90 Degree Scatter

Neutrophils

Lymphocytes

Monocytes

Forward Scatter

Human white blood cells

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Different size cells

Particle or cell size (log scale)

Number of events

small

large

0.1 1 10 100 1000

0.9

20

90

200

700

While forward light scatter is not always related to cell size, in

The majority of cases between 1
-
20 microns, it is a reasonable estimate

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Light Scatter of white blood cells


Light scatter can be used to identify
populations of cells

x

In peripheral blood, the three
main populations of leukocytes
can be distinguished. A “gate” or
“bitmap” can be placed around a
region so that further analysis
can be made on this region. The
cells in the region marked “X”
can be evaluated as a population.

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Fluorescence
-

e.g. Monoclonal
Antibodies

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

“B” Cells

“T” Cells

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

3 Parameter Data Display

Isometric Display

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

The Cell Cycle

G
1

M

G
2

S

G
0

Quiescent cells

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Definitions & Terms


Ploidy


related to the number of chromosomes in a cell


Haploid
: Number of chromosomes in a gamete (germ cell) is called the
HAPLOID number for that particular species


Diploid
: The number of cells in a somatic cell for a particular species


Hyperdiploid
: greater than the normal 2n number of chromosomes


Hypodiploid
: Less than the normal 2n number of chromosomes


DNA Tetraploidy
: Containing double the number of chromosomes


DNA Index
: The ratio between the mode of the relative DNA content of
the test cells (in G
0
/G
1
phase) to the mode of the relative DNA content in
normal G
0
/G
1

diploid cells


Coefficient of Variation
-

CV
: The ratio between the SD of the mode of
the G
0
/G
1

cell populations expressed as a percentage.


Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Normal Cell Cycle

G
0

G
0

-

G
1

s

G
2

M

DNA Content

2N

4N

G
2

M

G
0

G
1

s

0


200


400


600


800

1000

0

75

150

225

300

Cell Count

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

A typical DNA Histogram

G
0
-
G
1

S

G
2
-
M

Fluorescence Intensity

# of Events

2n

4n

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Flow Cytometry of Apoptotic
Cells

PI
-

Fluorescence

# Events

Apoptotic cells

Normal G0/G1 cells

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Analyzing the DNA Histogram

DNA Content
0
50
100
150
200
Number
0
200
400
600
Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Chromosome Analysis

Most human chromosomes can be separated by flow cytometry

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Chromosome Analysis


(Bivariate

Flow Karyotyping
-

porcine)

chromosome 1

chromosome 2

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Ethidium

PI

600 nm

300 nm

500 nm

700 nm

400 nm

457

350

514

610

632

488

Spectra of PI and EtBr

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

log Thiazole Orange

.1


1000


100


10


1

Count

0


150


112


75


37

RMI = 0

log Thiazole Orange

.1


1000


100


10


1

Count

0


150


112


75


37

RMI = 34

Reticulocyte Analysis

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

4 colors
-

simultaneous collection

(can go to 17 colors)

Emission wavelength (nm)

530


580

630


680 730 780

FITC

PE

PE
-

TR

PE
-
CY5

We separate different subsets by taking bands of light from the

light spectrum and analyzing the intensity of light in that band

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

10
1
10
2
10
3
10
4
CD56 -->
10
1
10
2
10
3
10
4
CD4 -->
10
1
10
2
10
3
10
4
CD3 -->
10
1
10
2
10
3
10
4
CD4 -->
CD3

CD3

CD3

10
1
10
2
10
3
10
4
CD3 -->
10
1
10
2
10
3
10
4
CD56 -->
10
1
10
2
10
3
10
4
CD3 -->
10
1
10
2
10
3
10
4
CD8 -->
CD56

10
1
10
2
10
3
10
4
CD56 -->
10
1
10
2
10
3
10
4
CD8 -->
CD56

CD8

10
1
10
2
10
3
10
4
CD8 -->
10
1
10
2
10
3
10
4
CD4 -->
FOUR COLOR PATTERN

CD4

CD8

CD56
-

NK

CD8

CD4

CD4

Data from Dr. Carleton Stewart

CD56


NK Cells

CD3


T cells

CD4


T cells


Helper

CD8


T cells
-

Cytotoxic

This is a subset of cells

It is CD3
+

CD56
+

This is a subset of cells

It is CD3
+

CD4
+

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Multicolor Analysis


Roederer, et al

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Cellular

Response:



Cell death



Cell ‘suicide’



Ignore damage



Damage repair



Incorrect repair

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Functional Assays


intracellular pH


intracellular calcium


intracellular glutathione


oxidative burst


phagocytosis

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Oxidative Burst


generation of toxic oxygen species

by phagocytic cells


superoxide anion measured

with hydroethidine


hydrogen peroxide measured with

2’,7’
-
dichlorofluorescin diacetate

(DCFH
-
DA)

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor



TIME (seconds)

0


2400


1800


1200


600

Log DCF
.1
1000
100
10
1
Scale

345

115

38

12

4

Neutrophil Oxidative Burst

PMA
-
Stimulated

Neutrophils

Unstimulated

Neutrophils

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

FITC
-
Labeled Bacteria

Phagocytosis

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Cellular Functions


Cell Viability


Phagocytosis


Organelle Function


mitochondria, ER


endosomes, Golgi


Oxidative Reactions


Superoxide


Hydrogen Peroxide


Nitric Oxide


Glutathione levels



Ionic Flux Determinations


Calcium


Intracellular pH




Membrane Potential



Membrane Polarization



Lipid Peroxidation

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Organelle Function


Mitochondria




Rhodamine 123


Endosomes




Ceramides


Golgi





BODIPY
-
Ceramide


Endoplasmic Reticulum


DiOC
6
(3)
Carbocyanine

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Fluorescent Indicators

How the assays work:


Superoxide
:
Utilizes hydroethidine the sodium borohydride reduced
derivative of EB


Hydrogen Peroxide
:
DCFH
-
DA is freely permeable and enters the
cell where cellular esterases hydrolyze the acetate moieties making a
polar structure which remain in the cell. Oxidants (H
2
O
2
) oxidize the
DCFH to fluorescent DCF


Glutathione
:

In human samples measured using 40

M
monobromobimane which combines with GSH by means of
glutathione
-
S
-
transferase. This reaction occurs within 10 minutes
reaction time.


Nitric Oxide
:
DCFH
-
DA can indicate for nitric oxide in a similar
manner to H
2
O
2
so care must be used. DAF is a specific probe
available for Nitric Oxide

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Hydroethidine

HE






EB

N

CH
2
CH
3

NH
2

H
2
N

H

Br
-

N

CH
2
CH
3

NH
2

H
2
N

+

O
2
-

Phagocytic Vacuole

SOD

H
2
O
2

NADPH

NADP

O
2

NADPH Oxidase

OH
-

O
2
-

DCF

HE

O
2
-

H
2
O
2

DCF

Example: Neutrophil Oxidative Burst

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

DCFH
-
DA DCFH
DCF

COOH

H

Cl

O

O
-
C
-
CH3

O

CH3
-
C
-
O

Cl

O

COOH

H

Cl

OH

HO

Cl

O

COOH

H

Cl

O

HO

Cl

O

Fluorescent

Hydrolysis

Oxidation

2’,7’
-
dichlorofluorescin

2’,7’
-
dichlorofluorescin diacetate

2’,7’
-
dichlorofluorescein

Cellular Esterases

H
2
O
2

DCFH
-
DA

DCFH
-
DA

DCFH

DCF

H O


2 2

Lymphocytes

Monocytes

Neutrophils

log FITC Fluorescence

.
1


1000


100


10


1

0

20

40

60

counts

PMA
-
stimulated PMN

Control

80

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Phagosome

O
2

O
2
-

H
2
O
2

NADPH + H
+

NADP
+

HMP

NADPH

Oxidase

GSSG

GSH

GR

GP

H
2
O
2

SOD

O
2
-

H
+

H
2
O

Catalase

H
2
O + O
2

PCB

SOD

PCB

(Reduced GSH level)

Stimulant

PKC

PCB

(PMA)

Human Neutrophil

?

?

+

O
2
-

OH
.

Lipid Peroxidation

Phospolipase A2 activity

Leukotrienes

H
2
O
2

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Hydroethidine

Superoxide Production

15 minutes

45 minutes

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Cell Sorting


Physically separating cells based on some
measurable characteristic


Placing these cells into containers


488 nm laser

+

-

Fluorescence Activated

Cell Sorting

Charged Plates

Single cells sorted

into test tubes

FALS Sensor

Fluorescence detector

Purdue University Cytometry Laboratories

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

SMALL BEAD

LARGE BEAD

Frequency Histogram

SMALL BEAD

LARGE BEAD

Sample in

Sheath

Sheath in

Laser beam

Stream

Charge

+2KV

-
2KV

Waste

SORT RIGHT

SORT LEFT

SORT DECISIONS

Piezoelectric

crystal oscillator

Last attached

droplet

LEFT

RIGHT

Sensors

Sensor

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Cell Sorting

Video of the droplet formation in a sort stream from a Cytomation
instrument.
Source: Purdue CDROM vol 4, 1998

Video2.mpg

+++

---

Purdue University Cytometry Laboratories


J. Paul Robinson, Professor

Lab

2 Groups of 8 students each

Hansen Hall, Room B50 (Basement)

Meet with Kathy Ragheb and Cheryl Holdman

One on April 18, 12:30
-
4:30

Other April 25, 12:30
-
4:30


Work in groups of 2 and you will take blood


Phenotype your own blood for T cell and B cells


Blood film and total blood count


Coulter count to obtain total cell numbers


Look at T and B cells under fluorescence scope