Download Flow Cytometry

Flow Cytometry
www.le.ac.uk
Applications
• FRET- protein interaction • Cell sorting (FACs)
• Membrane protein
expression
• Intracellular pH
• Intracellular protein
expression
• Cell proliferation
• Cell viability
• Ca2+ flux
• Cell cycle analysis
• RNA content
• Production of
intracellular oxidative
species
General principles
• Fluidics
• Optics
• Detection
• Output
Fluidics
• Sample uptake
– Older machines under vacuum
– Newer machines inject sample
– Under high pressure
• Injected into centre of a flow chamber
• Buffer or dH2O (Sheath fluid) flow round the sample causing
cells to form single line
– Hydrodynamic focusing
Optics
• Samples move through path of laser as single cells
– Different types of laser
•
•
•
•
Violet laser – 405nm
Blue laser – 488nm
Yellow laser – 561nm
Red laser – 635nm
– Numbers represent wavelengths at which the lasers excite
Fluorophore's
– The blue laser is also used to measure size (forward
scatter) and granularity of cells (side scatter)
Optics continued…
• Laser light reflects off cells and reaches dichroic mirrors
– Mirrors allow certain wavelengths through and all other
wavelength's are reflected to detectors
– There are three types of filter
• Long pass – allows light through over a certain wavelength
–
E.g. 750LP- light >750
• Short pass – allows light through below a certain wavelength
–
E.g. 560SP- light <560nm
• Bandpass – allows light through between certain wavelengths
–
E.g. 530/20 – light between 520 and 540nm
• The fluorophore’s you are able to use are dependent on the filters you
have – and their emission wavelength
Optics continued…
Detection
• Detected light hits photomultiplier tubes (PMTs)
• Creates voltage pulse proportional to amount of light they receive
– E.g. small cell, low forward scatter, small pulse
– Large cell, high forward scatter, large pulse
• Voltages from fluorescent signals converted to numerical values
Output
• Data collected represented in graphical format
• Histogram, dot plot, contours…
Important considerations
• Fluorophore’s / Lasers
• Location of molecule of interest
• Controls
– Isotype
– Secondary control
– Positive
• Compensation
• Gating
• Method
Fluorophore’s
• APC-H7
• PE-Cy7
• VioGreen
• APC-Cy7
• PE-Cy5.5
• AmCyan
• APC-Vio770
• PE-Cy5
• Pacific Blue
• APC
• PE-Texas Red
• VioBlue
• PerCP-Cy5.5
• PE
• PI
• PerCP
• FITC
• Alexa Fluors’s
• PE-Vio770
• Pacific Orange • Lots more
Fluorophore’s continued…
• The Fluorophore's you can use depend on your lasers and filters
• E.g. FITC (green) Excited 495nm, emission 519nm
– Stokes shift
– Use 488 blue laser and 530/40 band pass filter
Fluorophore’s
Controls-1
• Isotype
– Antibody from the same species as host and same
antibody isotype
• E.g. we have a mouse IgG2a anti-CD14-FITC antibody so
an isotype would be mouse IgG2a-FITC, usually used at
same concentration under same conditions
• Allow a measure of non-specific binding
Controls-2
• Sometimes primary antibody to molecule of interest isn’t
labelled
• Have to use labelled secondary antibody to detect primary
• Standard procedure to use a secondary only sample as
control
Controls-3
• Positive controls
– Need to know if antibody works
– Optimum concentration of primary antibody
– Best method to stain
Compensation
• If using more than one fluorophore
• Needed if Fluorophore's excited by same laser
• Fluorophore’s have emission spectra
– Values given are peak emission values
• In the real world emission spectra are graded
Compensation continued…
• Some emission spectra of fluorophore’s overlap
• Means that they will fluoresce in that channel
• If no compensation than you would not know if your
result is real
• Most new machines now have automatic settings
Where is your molecule of interest?
• Membrane
– Staining relatively simple no fixation or
permeabilisation required
• Cytosol/organelle
– Permeabilisation and fixation required
• Protein? DNA? Phospholipid?
• Antibodies need to be able to enter a cell
• Some dyes don’t e.g. Calcein
Methods
• Each lab has standard methods
• Usually results do not differ much between similar methods
• However, different methods need to be used if protein
intra/extracellular
– Permeabilisation
• Acetone / Tween / Triton
– Fixation
• Methanol / PFA / Formaldehyde
• Also need to lyse RBCs if using whole blood
Example
• Protein expressed on surface of endothelial cells
• Cultured Ea.Hy926 cells
– Know contain protein by western blotting
Conventional method
• Resuspend cells in buffer
• Stain 30min primary antibody
• Stain 30min secondary antibody
• Make up to 1ml in buffer
• Run on flow cytometer
Adjusted method
• Cells fixed for 5min in methanol
• Resuspended in buffer
• Stained as before
Gating
• Gates can be used:
– To select populations
– To measure the level of protein expression
• E.g. in whole blood can gate monocytes based on size and granularity
Monocytes
Granulocytes
Lymphocytes
Gating continued…
• Can set histogram to identify molecule of interest on
gated cells
Gating continued…
• When measuring level of protein expression etc. the
isotype is usually set to 2%
• To take into account cells that express the molecule
of interest weakly
Stats
• Can get a number of statistics from the data
• Most useful
– Percentage of positive cells
– Median fluorescence
• Intensity and spread of distribution
Worked example
PE
FITC
Compensation
Compensation
10 4
R4
10 4
R5
Isotype
10 2
10 1
10 0
100
R7
101
R4
102
FITC Log Comp
R6
103
10 0
100
104
10 4
R5
10 3
10 2
4h
10 1
101
R4
102
FITC Log Comp
103
104
103
104
R5
10 2
8h
10 1
R6
10 0
100
R7
10 3
PE Log Comp
PE Log Comp
0h
10 2
10 1
R6
10 4
R5
10 3
PE Log Comp
PE Log Comp
10 3
R4
R7
101
102
FITC Log Comp
R6
103
104
10 0
100
R7
101
102
FITC Log Comp
Facilities in the department
• Currently 2 flow cytometers
• Both Beckman Coulter
– Gallios
– ADP Cyan
MUST BE TRAINED AND
HAVE INDUCTION FROM
TINA JAMES BEFORE YOU
START USING EITHER
MACHINE!!
Gallios
• 3 lasers, 488 (blue), 638 (red), 405 (violet)
• Filters allow up 10 colours
– FL1 525/40 bp
– FL2 575/30 bp
Blue
– FL3 620/30 bp
– FL4 695/30 bp
– FL5 755 LP lp
– FL6 450/40 bp
Violet
– FL7 550/40 bp
– FL8 660/20 bp
– FL9 725/20 bp
Red
– FL10 755 lp
• Brand new
• Carousel so can load and leave
• Kaluza software, easy to use
ADP Cyan
•
3 lasers, 488 (blue), 405 (violet) and 635 (red)
– Although red filter temperamental and not always aligned
•
Filters allow up 9 colours
– FL1 530/40 bp
– FL2 575/25 bp
– FL3 613/20 bp
Blue
– FL4 680/30 bp
– FL5 750 LP
– FL6 450/50 bp
Violet
– FL7 530/40 bp
– FL8 665/20 bp
Red
– FL9750 LP
•
•
•
No carousel so have to manually load samples
Software easy to use and transferable to work computer
Can add gates, analyse and set up histograms after
running samples
Documentation
• Keeney, M., et al., Isotype controls in the analysis of lymphocytes and
CD34+ stem and progenitor cells by flow cytometry - Time to let go!
Cytometry, 1998. 34(6): p. 280-283.
• http://www.beckmancoulter.com/wsrportal/wsr/research-anddiscovery/products-and-services/flow-cytometry/flow-cytometers/cyanadp-analyzer/index.htm - For CYAN ADP guide
• http://www.biolegend.com/spectraanalyzer
• Flow Cytometry- a basic introduction, Michael G. Ormerod
• Most of the companies have information on flow cytometry
• I also have 4/5 power points and training manuals from Beckman Coulter