Transcript Fundamentals and Applications of Flow Cytometry Scott Tighe Flow Cytometry Core Lab at the Vermont Cancer Center HSRF 305 656-2557

Fundamentals and
Applications of Flow
Cytometry
Scott Tighe
Flow Cytometry Core Lab at the Vermont Cancer Center
HSRF 305
656-2557
Overview
• Basics components of a cytometer
• Fundamentals of photonics-optics and
fluorescence
• Software and modeling
• Types of analysis
• Sorting for cells and RNA
• Sample requirements-[controls, compensation, titer
Ab, blocking]
• Sign-up for time on VCC instruments
What is Flow Cytometry?
An instrument for making cell-based fluorescent measurements.
Method for quantitating cellular or structural components of a cell
using fluorescent antibodies or probes.
Allows Analysis of tens of thousands of cells in minutes.
A method to sort and collect specific cell types.
The BD FACS ARIA with sorter
Beckman Coulter Epics XL
Hardware Components of Flow Cytometer
Fluidics- Cells are carried to laser in a saline-based sheath fluid.
Pneumatics-Pressure drives the fluid flow
Optics- laser, band-pass filters, and PMT detectors.
Computer-Performs the analysis
Older MoFlo cytometer by Cytomation
Fluidics
THE FLOW CELL
Laser focusing and
hydrodynamic focusing
LASER
Waste
Tank
Sheath
Tank
Vacuum
Sheath
Pressure
(Constant)
Sample
Pressure
Line Pressure
The Flow cell:
showing hydrodynamic focusing
Higher the sample flow the wider the
sample stream and lower the resolution
Fluorescence
signals
Focused laser
beam
Injector
Tip
Sheath
fluid
Excitation Sources
Lets consider two items
Fluorochrome
The electromagnetic spectrum
excitation-emission curve
Lasers
• Lasers provide Coherent Light (Single mode-single wavelength)
• High Power
• Narrow band width
• Can be tunable or fixed wavelength
• Gas tube or Pumped solid state-which allows coverage for all fluorochromes!
GAS
Krypton-647nm
Argon -488nm
HeNe [633nm]
PSS
Nd-Yag (AlGaAs diode or Krypton lamp pumped)
Freq. Tripling !!! 1064,532,355nm
Great for Flow cytometry
UV-364nm [from Ar]Ti:sapphire (Ar pumped) 650-1100 nm
Excimers [193-284nm]
This is just a few….
Output frequencies of common tunable gas tube lasers
Light Emitting Diodes [LED]
-Benefits
-Cons
– Cheaper
– Smaller
– Easily Available
-Not as bright
– Many wavelengths
-Not available for UV
-Broad excitation spectra
Fluorescence and Photons
Fluorescence
• the molecular absorption of a photon triggers
the emission of another photon with a longer
wavelength.
Ext. coef + Quantum yield is
Quantum efficiency
Stoke shift
Release of
Photon
The fluorochrome: Phycoerytherin
488 nm Argon
575 nm
Large multi-subunit, globular (~240 kDa)
Excitation 488 nm
Emission Maximum 575 nm
>20 chromophores per molecule
High quantum yield (bright)
Optics and Detection
Detecting and sorting of different colors from cellular emission
involves band pass filter and beam splitters
Laser (Argon 488nm)
Freq
Freq
FS Sensor
Red Signal
Green Signal
Fluorescence detector
(PMT1, PMT4 etc.)
Optics in a Flow Cytometer
Beam
Splitters
Forward Angle [FS] Light Scatter
Provides data on size- The bigger the cell, the larger the FS
Laser
FS Sensor
[size]
Side Scatter [SS] Detector
Provides data on internal structures
The more structures, the more ss
Granulocytes have high ss
Provides data on surface characteristics
Dead cell have a rougher cell surface and a higher ss
RBC have little to no ss
Laser
FS Sensor
SS Sensor
[granularity]
Beam Splitters
Dichroic Filter/Mirror at 45 degrees
Laser Light+
Sample Light
488-680nm
BS 625
500-680nm
500LP
500-624 reflected
Reflected light
Transmitted Light
625nm and above
Band Pass Filters
630/30nm BandPass Filter
White Light Source
Transmitted Light
A 630/30 BPF will only allow 615-645nm through
Long Pass Filters
Light Source
520 nm Long Pass Filter
>520 nm
Light
Short Pass Filters
Light Source
575 nm Short Pass Filter
<575 nm
Light
Note:
Great for microscopes
Detectors
Photomultiplier tubes (PMT)
Photodiode (PD)
“Old” but good technology
New[er] technology, still not common
Most common detector used in flow
High quantum efficiencies for visible
High sensitivity but poor quantum efficiencies
in red (>650nm)
No internal gain adjustment
Able to adjust gain to over 100,000
Common
Inexpensive
Requires Cooling
Require a high voltage bias
Flow cytometers may have 4 to 6 detector channels [or more]
PMT
4
Bandpass filter
PMT
Dichroic
3
filters
PMT 2
PMT 1
Flow cell
Laser
PMT=Photomultiplier Tubes-do not see colors, only photons.
Spectral Compensation
Must be performed when using fluorochromes with overlapping
emissions
Sorting Cells
The Cytomation Mo Flo cell sorter
Analyzes and sorts cells at 70,000 cells per second
Cost $ 350,000
Sorting
488 nm laser
FS Sensor
Fluorescence detector
Charged Plates
Single cells sorted
into test tubes
-
+
Data Output
Histograms
Data Output is represented by a histogram
-Single parameter trace [one color]
Fluorescent intensity-
-Dual paramater dot plot [two color]
Gating
Allows the ability to select specific cell populations in one
histogram and analyze for additional parameters [colors] in
additional histograms
Comparison of single and dual
parameter histograms
Specific Types of Analysis Done
Using Flow Cytometry
Some Typical Applications of Flow Cytometry?
Immunophenotyping
DNA cell cycle/tumor
ploidy
Membrane potential
Ion flux
Cell viability
Karyotyping
Cell tracking and
proliferation
Sorting
Redox state
Chromatin structure
Cell proliferation assay
Cell enumeration and sizing
Apoptosis
Phagocytosis
Intracellular pH
Intracellular calcium
Oxidative burst
Intracellular antigen measurement
Cytokine detection
Reticulocyte analysis
Platelet analysis
Immunophenotyping
Immunophenotyping
Classifying immune cells using cell surface antigens
CD4
CD3
CD3-T-cell
CD4-T helper
CD # = cluster designation number
The structure of IgG-An Antibody
Fab
regions
Fab
regions
Fc receptor
Two Types of Antibody Labeling
Direct labeling: Uses one antibody that has a fluorochrome conjugated
directly on it. One step staining. Easier.
Indirect labeling: Uses two
antibodies.The first is “against” a
specific antigen on the cell. The second
antibody is fluorochrome-labeled and is
“against” the first. More complicated.
Cell
Cell
Example of Data
.1
1
10
Using two mABs with dyes of different color outputs
.1
1
10
100
1000
Non-Specific Antigen Blocking
Blocking is important to avoid false positives
Non-specific binding of antibodies is really Fc binding
Typically a serum source (BSA, FCS) is used but is often
not adequate.
Recommend goat IgG at 100-200ug/ml
mAB Titering-A must!!!!
Uses a specific Number of cells against antibody dilutions.
Perform on new lots of antibodies
Cell Cycle
Cell Cycle Analysis
Indicates the rate and stage of cell replication or division.
Propidium Iodide most common dye.
The dye intercalates into the DNA strand.
S Phase
(Synthesis)
s
C
o
u
n
t
0
75
G1
150
225
G0
Counts
G2
M
0
200
400
2N
G1/G0
600
800
4N
G2M
1000
Modeling Cell Cycle data
When peaks are close together and overlapping, it is important to use
specific software to model the data and get accurate results
ModFit 3D 3.0
WinList 5.0
FloJo
WinMDI is a free software written by J.Trotter and is available on the web.
Cell Cycle of GFP Cells
Cell Viability
Cell Viability
Simplest method using cell permeabilization
-Propidium Iodide
-7AAD
-Sytox
Membrane potentials are a good indicator such
mitochondria membrane potential [JC1].
Redox dyes-DHF, DHR- Turn colorless by reduction in cells.
Enzyme activity probes-Esterase activity using cFDA, cell tracker
dyes, calcein,… converted to fluorescent probe by enzymes.
Cell Viability using Dye Exclusion-Propidium Iodide
How the assay works:
•
•
•
PI cannot normally cross the cell membrane
If the PI penetrates the cell membrane, it is assumed to be damaged
Cells that are brightly fluorescent with the PI are damaged or dead
Dead
Live
PI
PI
PI
PI
PI
PI
Pi fluoresence >>>
PI
PI
PI
PI
PI
PI
PI
PI
Apoptosis
Apoptosis-Annexin staining
Uses Annexin-FITC against Phosphatidylserine to determine membrane translocation
Uses PI to determine membrane permeability
Must be used cautiously on adherent cells do to trypsin
Not for fixed cells
Apoptotic
Necrotic
Annexin
Necrotic
PI
APO-BRDU-TUNEL
DNA fragmentation
Available 3’OH
Uses Tdt to add BrDU
Stain with anti-BrDU
Proliferation Assays
Proliferation can be measured by cell cycle, Brdu incorporation,
and membrane dyes such as CFSE and PKH26.
Brdu incorporation to S-phase DNA
CFSE, PKH 26,
Sorting cells for RNA
Decon the flow cytometer with bleach
Run a test on dummy cells first
check your viability
Ensure all reagents are RNase-free
Sort into an extraction buffer when applicable
or sort into sterile media
Extract RNA on the same day if in an extraction buffer
*****Please come get a handout at our lab regarding these
protocols
Sample requirements
• Negative no stain controls are required for most runs
• Single color controls are required for spectral compensation when
performing two or more color analysis
• Samples should be in 12x75 plastic tubes containing 800ul of cell
suspension at a concentration of 100,000-1 million cells
• Cells that are clumpy must be filter thru 70um mesh
• If using dual antibody staining, a secondary only will be necessary as
well as your no stain control
• All analysis types should be accompanied with a positive control
sample in order to validate the staining, protocol, and compensation
Logging into the VCC Flow cytometer
Billing and sign up is done using the BioDesktop
DNA facility>>>Shared Inst. Sign-up>>>Flow cytometer
Reserve the time you plan to use it >>>OK
Enter your contact info
Enter your chartstrings into the chartstring manager
You may edit after your run
An email will also be sent to you in case you need to edit your time for
billing purposes
Please sign up in the log-in book as well
The new rate for the VCC flow is $9.51 [cancer] and 19.01 [non-cancer] per ½ blocks
Thank you for your time