Multi-parameter Flow Cytometry: available dyes and combined usage

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Multi-parameter Flow Cytometryavailable dyes
and combined usage
Imperial College London
Martin R. Goodier m.goodier_at_ic.ac.uk Departme
nt of Immunology Imperial College London
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Multi-parameter flow cytometry

• Permits the detailed analysis of markers of
  cellular differentiation ..uses in
  developmental systems and in the assesment of
• vaccination-driven responses.
• Permits the simultaneous evaluation of cell
  phenotype and function

Surface phenotype
Cell cycle DNA RNA
Factor production
Signal transduction Intracellular calcium pH
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Considerations when choosing dye combinations

• Available light sources for dye excitation
• Potential overlap/ interaction between different
  dyes

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Laser Emission in the UV/ Visible spectrum
Wavelength nM
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Absorbtion spectra of some commonly used dyes
Excitation wavelength
FITC R-PE APC
650nM
495nM
488/565nM
Absorbance
400 500 600 700
Wavelength nM
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Dyes used for phenotypic analysis and excitation
wavelength
588nM Rhodamine Texas Red Cascade
yellow Cy5.5-APC (APC) (APC-Cy7)
488nM FITC Alexa Fluor 488 R-PE PE-Texas Red
(ECD) PE-Cy5 PerCP PerCP-Cy5.5 PE Cy7
647 nM APC Alexa Fluor 647 APC-Cy7
405nM Alexa Fluor 405 Alexa Fluor 430 Cascade
Blue (Cascade Yellow)
Some dyes (..) can be activated over a broad
range of different excitation wavelengths
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DNA, RNA and cell viability measurements
UV Hoescht 33342 (DNA) DAPI (DNA)
488 nM Acridine Orange (DNA) Acridine Orange
(RNA) Propidium Iodide (DNA) 7-AAD (DNA)
588nM Ethidium monoazide Pyronin Y
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Cell function dyes
633nM SNARF (pH)
UV Indo-1 (Ca2)
488 nM Fluo-3 (Ca2) Dioxycyanin Rhodamine 123
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Emission spectra of commonly used dyes
FITC R-PE ECD APC PE-Cy7
525nM 578nM 613nM 660nM 760nM

Emission
500nM 600nM 700nM
800nM
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Spectral overlap is detected by different
photo-multiplier tubes
Photo-multiplier tube
FL-2 PMT
FL-1 PMT
FL-3 PMT
FL-4 PMT
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Electronic compensation
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Achievable number of parameters (Modified BD
FACStar plus)

• T- cell differentiation naïve T-cells

Excitation Dye Marker
Cascade Blue Cascade Yellow FITC PE Cy5PE Cy5.5PE
Cy7PE
CD45RA CD3 CD28 CD49f CD11a CD27 CD4 CD62L CD45R
O CD8 CD57
405nM
488nM
Forward Scatter Side Scatter 13 parameters
595nM
Alexa595 APC Cy5.5-APC Cy7-APC
De Rosa et al, Nature Medicine 2001
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Achievable parameters
2. Intracellular signalling
Excitation Dye Marker
Cascade Blue Cascade Yellow ALEXA 546 ALEXA
568 Cy5PE Cy5.5PE Cy7PE
CD11a CD8 JNK P-AKT-ser473 EMA CD4 CD62L p-TYK2
p-JNK CD28 CD45RA
405nM
488nM
Forward Scatter Side Scatter 13 parameters
595nM
Alexa595 APC Cy5.5-APC Cy7-APC
Perez and Nolan , Nature Biotechnology 2002
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References

• Panchuk-Voloshina N. et al Alexa dyes, a series
  of new fluorescent dyes that yield exceptionally
  bright, photostable conjugates. J Histochem
  Cytochem. 1999 Sep47(9)1179-88.
• Berlier JE et al Quantitative comparison of
  long-wavelength Alexa Fluor dyes to Cy dyes
  fluorescence of the dyes and their bioconjugates.
  J Histochem Cytochem. 2003 Dec51(12)1699-712.
• Comparisons of the performance of Alexa dyes
  with other fluorochromes.
• Perez O.D. and Nolan G.P.Simultaneous
  measurement of multiple active kinase states
  using polychromatic flow cytometry. Nat
  Biotechnol. 2002 Feb20(2)155-62.
• De Rosa S.C. et al 11-color, 13-parameter flow
  cytometry identification of human naive T cells
  by phenotype, function, and T-cell receptor
  diversity.Nat Med. 2001 Feb7(2)245-8.
• Potential application of multiparameter
  (polychromatic) flow cytometry.
• E. mail m.goodier_at_imperial.ac.uk