Technical Seminar

1
Technical Seminar
FLOW CYTOMETRY AND CELL SORTING
Bill HyunJane GordonSarah Elmes
2
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

3
LCA/LBNL Flow Cytometers

• FACScan
• 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

4
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

5
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

6
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

7
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

8
Fluorescent Cell Properties
9
Optical Design
PMT 5
PMT 4
Sample
PMT 3
Dichroic
Filters
Flow cell

PMT 2
PMT 1
Scatter
Sensor
Bandpass
Laser
Filters
10
Scatter Pattern of Human leukocytes
A flow cytometry scattergram
Forward scatter (size)
Side scatter (granularity)
11
Flow cytometry measurements
SCATTER FLUORESCENCE IMAGE
G
M
L
12
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
13
Fluorescence

• Stokes Shift
• is the energy difference between the lowest
  energy peak of absorbance and the highest energy
  of emission

Stokes Shift is 25 nm
Fluorescein molecule
520 nm
495 nm
Fluorescnece Intensity
Wavelength
14
Common Fluorescent Probes
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
15
Fluorescence signal separation
16
Spectral Overlap
17
Compensation
FITC Beads AloneNo Compensation
Three Beads
Electronically compensated display
18
Fluorescence

• Resonance Energy Transfer

Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Intensity
Absorbance
Absorbance
Wavelength
19
Fluidics
20
Cell Sorting
Sample in
Sheath
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).
Stream Charge
Piezoelectric crystal oscillator
Sheath in
Sensors
SORT DECISIONS
Laser beam
SMALL BEAD
LARGE BEAD
Sensor
SORT RIGHT
SORT LEFT
Last attached droplet
Frequency Histogram
-4KV
4KV
RIGHT
LEFT
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.
Waste
SMALL BEAD
LARGE BEAD
21
SORTING
22
Data Presentation Formats

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

23
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

24
Data Representation
25
Chromosome ID and Sorting
26
The Cell Cycle
M
G2
G1
Quiescent cells
G0
S
27
A DNA histogram
G0-G1
Cell Number
G2-M
S
Fluorescence Intensity
28
A typical DNA Histogram
G0-G1
G2-M
S
of Events
Fluorescence Intensity
29
REtics
Reticulocyte Analysis
RMI 34
RMI 0
1000
1000
.1
100
1
10
1
10
100
.1
log Thiazole Orange
log Thiazole Orange
30
Labeling Strand Breaks with dUTP
Green apoptotic cells
R2 Apoptotic Cells
Green Fluorescence
Side Scatter
R1 Normal Cells
Red normal cells
PI-Red Fluorescence
Fluorescein-deoxyuridine triphosphate (dUTP)
Forward Scatter
Green Fluorescence
Green Fluorescence is Tdt and biotin-dUTP
followed by fluorescein-streptavidin Red
fluorescence is DNA counter-stained with 20µg/ml
PI
31
Three Color Lymphocyte Patterns
CD4
CD4
CD3
CD8
CD8
CD3
Data from Dr. Carleton Stewart
32
mixture
Run on Coulter XL cytometer
mixture
Scatter
BG
BG E.coli
E.coli
Scatter
Fluorescence
YoYo-1 stained mixture of 70 ethanol fixed
E.coli cells and B.subtilis (BG) spores.
33
Live cell/dead cell PI Fluorescence
PI
Hoechst 33342
Data from Dr. Doug Redelman, Sierra Cytometry
34
Oxidative Reactions

• Superoxide Hydroethidine
• Hydrogen Peroxide Dichlorofluorescein
• Glutathione levels Monobromobimane
• Nitric Oxide Dichlorofluorescein

35
Calcium Flux
Flow Cytometry
Image Cytometry
0.8
0.7
0.6
0.5
0.4
Ratio intensity of 460nm / 405nm signals
0.3
0.2
Stimulation
0.1
Time (seconds)
0
36
72
108
144
180
Time (Seconds)
0
0
50
100
150
200
36
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

37
Summary

• Main Applications
• DNA and RNA analysis
• Phenotyping
• Cell Function
• Sorting and cell isolation
• Immunological assays