U C S F cancer center Laboratory for Cell Analysis Technical Seminar FLOW CYTOMETRY AND CELL SORTING Bill Hyun Jane Gordon Sarah Elmes

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Laboratory for Cell Analysis

Technical Seminar

FLOW CYTOMETRY AND CELL SORTING

Bill HyunJane GordonSarah Elmes

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Laboratory for Cell Analysis (LCA)

• Over 450 Users of LCA shared instrumentation - More than 40 current clinical and research projects- LCA provides support to over $10 Million of annual UCSF extramural funding

• Cytometry support for Bay Area hospitals, biotech companies, research foundations, and other academic institutions

• Northern California Cytometry Group

• LCA Cytometry Courses – Image and Flow

• LCA Protocol Library

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LCA/LBNL Flow CytometersFACScan• 3 - color, 5 - parameter benchtop analyzer

FACS Calibur (three units)• 4 - color, dual laser (488 and 633nm)• Sample loader - walk away

FACS Vantage SE DIVA• 9-parameter, 3-laser optical bench• High speed sorting - rare event• Auto-cloning - 96 well plate• Index sorting and abort-save

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Technical Seminar

Principles of Flow Cytometry- Features- Flow Chamber- Lasers and Optics- Fluorescence and Light Scatter- Electronics- Data Processing

Data Acquisition and Handling- Philosophy- Data Analysis

Cell Sorting

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What is Flow Cyto/metry??

Flow cytometry is a technology that allows the SIMULTANEOUS MEASUREMENT OF MULTIPLE PHYSICAL CHARACTERISTICS OF A SINGLE CELL.

These measurements are made on a per cell basis at routine rates in a moving stream.

Mack Fulwyler, 1982

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Flow Cytometry – Partial History

• 1934 Moldavan – red blood cells measured in a microscope with a capillary flow and a photodetector

• 1945 Reynolds – laminar flow system• 1947 Coulter – patent for cell counter• 1964 Kamentsky – 500 cells/second measured for light

scatter and uv absorption• 1965 Fulwyler – electrostatic cell sorter based on volume• 1966 Van Dilla – sorting based on DNA content• 1969 Hulett et al – sorting based on cell fluorescence• 1972 BDIS – first commercial cell sorter• 1975 Gray – chromosome sorting

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What can flow cytometry do?

• Enumerate particles in suspension

• Evaluate 105 to 106 particles/cells in less than 1 min

• Measure particle-scatter as well as innate fluorescent

• Measure 2o fluorescence from multiple fluorochromes

• Sort single particles/cells for subsequent analysis or growth

• Separate “live” from “dead” particles

• Give operators white hair

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Fluorescent Cell Properties

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Optical DesignOptical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

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Scatter Pattern of Human leukocytes

Lymphocytes

Monocytes

NeutrophilsA flow cytometryscattergram

For

war

d sc

att e

r (s

ize)

Side scatter (granularity)

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Flow cytometry measurements

L

M

G

SCATTER FLUORESCENCE IMAGE

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Instrument Components

Fluidics: To introduce and restrict the cells for interogation. Controls specimen, sorting, rate of data collection

Optics: An excitation source and emission optics to generate and collect light signals. Light source(s), detectors, spectral separation

Electronics: To convert optical signals to proportionate electronic signals and digitize them for computer analysis. Control, pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control

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

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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

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Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488

Common Fluorescent Probes

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Fluorescence signal separation

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Spectral Overlap

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Compensation

FITC Beads AloneNo Compensation

Three Beads

Electronically compensated display

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Fluorescence

Resonance Energy Transfer

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

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Fluidics

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SMALL BEAD LARGE BEAD

Frequency Histogram

SMALL BEAD LARGE BEAD

Sample in Sheath

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.

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 introduced to surround the insertion rod causing hydrodynamic focusing 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).

Cell Sorting

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SORTING

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Data Presentation Formats

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

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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 during analysis

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FITC Fluorescence

Mo1

CD4 CD8

CD8

CD45

leu11a

CD20 Tube

ID

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Chromosome ID and Sorting

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The Cell Cycle

G1

MG2

S G0Quiescent cells

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A DNA histogram

G0-G1

S

G2-M

Fluorescence Intensity

Cel

l Num

ber

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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 REtics

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Labeling Strand Breaks with 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

[Fluorescein-deoxyuridine triphosphate (dUTP)]

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Three Color Lymphocyte Patterns

CD3CD4

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD4 -->

CD3

CD4

CD8CD8

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

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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 cytometer

Sca

tter

Fluorescence

Sca

tter

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Live cell/dead cell PI Fluorescence

Data from Dr. Doug Redelman, Sierra Cytometry

PIHoechst 33342

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Oxidative Reactions

Superoxide Hydroethidine

Hydrogen Peroxide Dichlorofluorescein

Glutathione levels Monobromobimane

Nitric Oxide Dichlorofluorescein

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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

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Membrane Potential

Oxonol Probes • Cyanine ProbesHow 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

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Summary

Main Applications

DNA and RNA analysis

Phenotyping

Cell Function

Sorting and cell isolation

Immunological assays

Documents

U C S F cancer center Laboratory for Cell Analysis Technical Seminar FLOW CYTOMETRY AND CELL SORTING Bill Hyun Jane Gordon Sarah Elmes