Transcript FITC

Introduction to the Principles
of Flow Cytometry
for users of the
Flow Cytometry Core Facility
at BUMC
21 September 2010
Mike Xie (X4-5225), [email protected]
Yan Deng (X4-5225), [email protected]
John Meyers (X8-7543), [email protected]
Gerald Denis (X4-1371), [email protected]
Flow Cytometry Core Facility: Personnel
Director of Cell Sorting Services: Yanhui Deng
Operations Manager: Mike Xie
Facility Consultant: John Meyers
Co-Director/Outreach: Gerald Denis
Director: David Sherr
http://www.bu.edu/cores/flow-cytometry
Where are the instruments located?
FACScan:
R901
FACScan,
FACSCalibur:L508
U R here
FACScan:
X620
LSR II,
MoFlo:670-5
http://www.bu.edu/cores/flow-cytometry
Flow cytometry was not easy in the old days
ungloved hands
missing PPE
gendered division
of labor
live anthrax
bacilli
But today…
it’s as easy as PCR.
fluidics
3 elements of any
flow cytometry system
electronics
optics
fluidics
sample
fluid
sheath
fluid
Fluidics
Purpose of the fluidics system:
1. Transport particles in a fluid stream
to the laser beam to be interrogated
2. Position the sample core in the center
of the laser beam
‘hydrodynamic focusing’
single file particles
● low flow rate
● high flow rate
● narrow sample
core
● wide sample
core
● high resolution
● low resolution
Always filter your samples to remove aggregates.
Fluidics
When conditions are right (i.e. when turbulence is minimal):
sample fluid flows in a central core
does not mix with the sheath fluid
This is termed ‘laminar flow’
“SSC”
“FSC”
electronics
optics
Forward and side scatter of leukemic cells
Malignant, large B cells
SSC
Normal B cells
FSC
The intensity of forward scatter light is proportional to
size and cross-sectional area of the cells.
The intensity of side scatter light is proportional to
size, shape and internal structure/irregularity of the cells.
Photomultiplier (PMT) detectors convert photons
(selected by mirrors and filters) to electrical pulses
Peak Height (volts)
Peak Area
Peak Width (time)
The higher the PMT voltage (user controllable), the greater
the output magnitude for a given photon. At higher PMT
voltages, the level of noise will also increase.
Photomultiplier Tubes (PMTs)
FSC: forward scatter
(size; cross-sectional
area)
SSC: side scatter
(granularity, internal
or surface structure
that scatters light)
Adjusting the voltage
of the PMT helps to
optimize capture
of desired populations
A dotplot represents two properties
of a single cell
250K
SSC-A
200K
150K
SSC
100K
50K
0
0
50K
100K
150K
FSC-A
200K
250K
FSC
A histogram represents the distribution
of a single parameter across many cells
10,000 cells each!
Control Condition
Experimental
100
% of Max
80
60
40
20
1 cell
1 cell
1 cell
1 cell
0
0
10
2
3
10
10
APC-A: pH2AX
4
10
5
Electronic processing of emission signals
Amplifiers are of two types: linear or logarithmic
Linear amplification is typically used with scatter.
Logarithmic amplification is typically used with fluorescence.
DNA content
(Linear detection)
DNA content
(Log detection)
Gating
250K
150
100
150K
# Cells
SSC-A
200K
100K
50
50K
0
0
0
50K
100K
150K
FSC-A
200K
250K
0
10
2
3
10
10
APC-A: pH2AX
4
10
5
Gating allows one to select populations based on computer or humanderived criteria and further gate or display the included cells
Backgating – don’t lose your bearings!
Backgating allows one to determine if a gating strategy is all-inclusive of a desired cell type.
In the above example, some cells are missed! What are they?
Many investigators overlook the importance of verification by backgating!
FLUORESCENCE
blue laser
(FITC)
Excitation wavelength and
emission wavelength are
unique properties of
each specific molecular
structure
Stokes Fluorescence
Excitation
Emission
Stokes shift
Laser light must overlap with excitation wavelength
Propidium iodide (PI)
488
488
Fluorescein (FITC)
ex
em
yes
em
488
Hoechst 33258
Texas Red
488
ex
no
ex
em
ex
em
Widely-used molecules are excited by
the 488 nm laser (FACScan)
488 nm (Blue) :
FITC, GFP, PE, PerCP, PE-Cy5, PI,
PerCP-Cy5.5, PE-Cy7
But different lasers are available to excite
other molecules (LSR II)
355
405
561
633
nm
nm
nm
nm
(UV) :
Indo-1, DAPI, Alexa Fluor 350, Hoechst 33258
(Violet) : Alexa Fluor 430, Alexa Fluor 405, Pacific Blue
(Yellow/Green): Texas Red, Cherry Red, Tomato Red
(Red):
APC, APC-Cy7, Alexa Fluor 647, Alexa 680
Octagon Detector Arrays
emitted fluorescent
light
longpass
dichroic
mirrors
bandpass
filters
EMISSION
Fluorescence detection
autofluorescence
weakly expressed strongly expressed
epitope
epitope
Note logarithmic scale
Isotype controls
How do you know
it’s real?
weakly expressed
epitope
isotype control test
Isotype control antibodies should be used at the same concentration to stain cells at the same
cell density as the experimental, but they give fluorescent signals that define a negative result.
Resolution sensitivity
Resolution sensitivity, the ability
to resolve a faint signal from
background) depends on the
difference D between the positive
and background peaks and the
spread of the background
peak W
Choose the right fluor for the job!
Reagent
Stain Index
Phycoerythrin (PE)
Alexa Fluor 647
356.3
313.1
APC
PE-Cy7
PE-Cy5
279.2
278.5
222.1
PerCP-Cy5.5
PE-Alexa Fluor 610
Alexa Fluor 488
FITC
PerCP
APC-Cy7
Alexa Fluor 700
Pacific Blue
AmCyan
92.7
80.4
75.4
68.9
64.4
42.2
39.9
22.5
20.2
i.e. pick a bright fluor for a
dim epitope
and avoid spillover of bright
cell populations into detector
channels that require high
sensitivity for rare signals
Problems in Emission Fluorescence
Excitation
Emission
Spectral overlap
Optical solutions to spectral overlap: Filters
Filters resolve overlapping wavelengths of emitted light
Longpass filter: transmits light of longer than or equal to
a specific wavelength
Shortpass filter: transmits light of shorter than or equal to
a specific wavelength
Bandpass filter: transmits light only within a narrow range
of wavelengths
Examples of optical filters in flow cytometry
Optical detector configurations
octagon
red trigon
APC
660/20
735 LP
bandpass
longpass
bandpass
EMISSION
two bandpass filters
Electronic solutions to spectral overlap: Compensation
To correct for emission spillover of FITC signal (normally detected
in the FL1 channel) into the FL2 channel (which detects PE),
it is necessary to use filters or electronic compensation or both.
Uncompensated
Optimal
COMPENSATION
Multicolor immunophenotyping
Before
After
No antibody. Autofluorescence only. No compensation applied.
CD4-PE. No compensation applied.
CD4-PE. Correct compensation applied.
1.4% PE subtracted from FITC PMT, 6.5% PE subtracted from APC PMT.
CD8-FITC. No compensation applied.
CD8-FITC. Correct compensation applied.
12.5% FITC subtracted from PE PMT.
CD4-PE + CD8-FITC. Streptavidin-APC alone.
CD4-PE + CD8-FITC. CD3-biotin + Streptavidin-APC
3 COLORS, CORRECTLY COMPENSATED
Spectral overlap of some fluorochrome combinations
cannot be compensated easily or at all
APC
Cy5
Therefore, avoid such combinations
Contour plots provide more accurate data
representation than dot plots
granularity →
Gates
size →
SSC
The uses of gates for cell sorting
CD8
FSC
CD3
Four Applications
Cell cycle analysis
Multicolor immunophenotyping
Phosphoprotein and kinase signaling
Stem cell sorting by the “side population” method
G0/G1
S
anti-BrdU-FITC
(DNA synthesis)
Cell cycle analysis
G2/M
S
G2/M
G0/G1
2N
2N 4N
DNA content (propidium iodide)
4N
7-aminoactinomycin D
Linear detection
Multicolor, Auto Compensation with Flow Jo
100
isotype
80
gate 1:
lymphocytes
% of Max
80
60
60
40
40
20
20
0
gate 2:
B cells
0
10 0
10 1
10 2
103
104
10
0
FITC
10
1
10
2
10
3
10
4
gate 6:
T cells?
Pacific Blue
isotype
100
isotype
100
80
10 4
80
% of Max
% of Max
% of Max
isotype
100
60
10 3
40
40
20
20
0
etc
60
gates 3 – 5
10 1
0
10
0
10
1
10
2
PE
10
3
10
4
10 2
10
0
10
1
10
2
10
PE-Cy7
3
10
4
up to 11 colors
10 0
0
200
400
600
800
1000
Filter configurations permit optimization
of multicolor stains
When two colors are not enough
A single PBMC sample simultaneously
stained with antibodies to quantify
expression of CD3, CD4, CD8,
CD7, CD27, CD28, CD45RA, CD62L
and CCR7.
A lymphocyte size gate and
CD3+/CD4-/CD8+ color gate is applied
to characterize stages of T cell
differentiation.
The T cell compartment cannot be
fully characterized by only 2 or 3
markers.
Current Protocols in Immunology 12:12 (2005)
Example of a 10-color experiment on our LSR II
Marker
Fluorochrome
B220: B lineage
Pacific Blue
IgM: mature B cells
PE-Cy7
IgD: mature B cells
PE
CD23: transitional B cells
FITC
AA4.1: transitional B cells
APC
Other markers
CD45.1: donor phenotype
PerCP-Cy5.5
CD45.2-Biotin: recipient phenotype
Streptavidin Alexa 350
CD11b/Mac-1: myeloid lineage
APC-Cy7
CD3: T lineage
Qdot 605
live/dead discrimination*
Invitrogen UV-activated vital dye
FITC Compensation
Matrix
FITC
FITC
“Phosphoflow” techniques allow you to measure
kinase cascades and signal transduction
Courtesy
John Meyers
[email protected]
Identification of a Hoechst 33342-staining
‘side population’ from murine bone marrow
Control
Verapamil
256
192
0.25%
192
FL5
FL5
blue
Hoechst
256
128
128
64
64
0
0
0
64
128
FL4
192
256
0
64
128
FL4
192
Hoechst red
100% of the gated side population is also Sca1+ ;
these are hematopoietic stem cells.
256
Kinetic identification of “side population” stem cells
These gated stem cells can be isolated by MoFlo
FCCF rates
On-line scheduling
Actual cytometer and work station use tracked to the minute,
recorded in and billed monthly by enterprise class central server
Unassisted
Assisted rate
Equipment rate (per hour) (per hour)
MoFlo
NA
$80.00
LSR II
$45.00
$67.50
FACScans
$40.00
$67.50
FACScalibur
$40.00
$67.50
Workstations
$10.00
NA
http://www.bu.edu/cores/flow-cytometry
Help and training:
Please sign up for basic training on FACScan or LSR2
with Yan Deng (X4-5225), [email protected]
Problems during experiments:
We can’t read minds. Please write a computer entry
in the COMPLAINT LOG for each instrument.
Or email us: [email protected]
We will contact you ASAP and usually can respond
within 2 hours.