Scott Tighe
Flow Cytometry Core Lab at the Vermont Cancer Center
HSRF 305 656-2557

• 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

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.
Beckman Coulter Epics XL
The BD FACS ARIA with sorter

Cells are carried to laser in a saline-based sheath fluid.
Pressure drives the fluid flow
laser, band-pass filters, and PMT detectors.
Performs the analysis
Older MoFlo cytometer by Cytomation

THE FLOW CELL
Laser focusing and hydrodynamic focusing
LASER
Waste
Tank
Vacuum
Sheath
Tank
Sheath
Pressure
(Constant)
Sample
Pressure
Line Pressure

Higher the sample flow the wider the sample stream and lower the resolution
Injector
Tip
Sheath fluid
Fluorescence signals
Focused laser beam

Lets consider two items
The electromagnetic spectrum
Fluorochrome 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
– Cheaper
– Smaller
– Easily Available
– Many wavelengths
-Cons
-Not as bright
-Broad excitation spectra
-Not available for UV

• 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)

Detecting and sorting of different colors from cellular emission involves band pass filter and beam splitters
Laser (Argon 488nm)
FS Sensor
Green Signal
Fluorescence detector
(PMT1, PMT4 etc.)
Red Signal

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]

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]

Laser Light+
Sample Light
Dichroic Filter/Mirror at 45 degrees
BS 625
Transmitted Light
500-680nm
488-680nm 625nm and above
500LP
500-624 reflected
Reflected light

White Light Source
630/30nm BandPass Filter
Transmitted Light
A 630/30 BPF will only allow 615-645nm through

Light Source
520 nm Long Pass Filter
>520 nm
Light
Note:
Great for microscopes
Light Source
575 nm Short Pass Filter
<575 nm
Light

Photomultiplier tubes (PMT)
“Old” but good technology
Most common detector used in flow
High sensitivity but poor quantum efficiencies in red (>650nm)
Able to adjust gain to over 100,000
Common
Inexpensive
Photodiode (PD)
New[er] technology, still not common
High quantum efficiencies for visible
No internal gain adjustment
Requires Cooling
Require a high voltage bias

Flow cell
Flow cytometers may have 4 to 6 detector channels [or more]
Dichroic filters
PMT
1
PMT 4
Bandpass filter
PMT 3
PMT
2
Laser
PMT=Photomultiplier Tubes-do not see colors, only photons.

Must be performed when using fluorochromes with overlapping emissions

The Cytomation Mo Flo cell sorter
Analyzes and sorts cells at 70,000 cells per second

Cost $ 350,000


488 nm laser
Charged Plates
-
Single cells sorted into test tubes
FS Sensor
Fluorescence detector
+

Data Output is represented by a histogram
-Single parameter trace [one color]
-Dual paramater dot plot [two color]
Fluorescent intensity-


Gating
Allows the ability to select specific cell populations in one histogram and analyze for additional parameters [colors] in additional histograms

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

Classifying immune cells using cell surface antigens
CD3
CD4 CD3-T-cell
CD4-T helper
CD # = cluster designation number

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

Using two mABs with dyes of different color outputs
.1
1 10 100 1000

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

Uses a specific Number of cells against antibody dilutions.
Perform on new lots of antibodies

Indicates the rate and stage of cell replication or division.
Propidium Iodide most common dye.
The dye intercalates into the DNA strand .
G
2
M
G
1 s t n
C o u
G
0
0
S Phase
(Synthesis)
200
2N
400
G1/G0
600
4N
G2M
800 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

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.

PI
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
Live
Dead
PI
PI
PI
PI
PI Pi fluoresence >>>
PI
PI
PI
PI
PI
PI
PI
PI

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
PI
Necrotic

DNA fragmentation
Available 3’OH
Uses Tdt to add BrDU
Stain with anti-BrDU

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,

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

• 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

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