Multi-parameter Flow Cytometry: available dyes and combined usage Martin R. Goodier Department of Immunology Imperial College London Imperial College London

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 Signal transduction Intracellular calcium pH Factor production

Considerations when choosing dye combinations Available light sources for dye excitation Potential overlap/ interaction between different dyes

Laser Emission in the UV/ Visible spectrum ULTRAVIOLET 300 RED ORANGE YELLOW GREEN BLUE VIOLET Wavelength nM UV Lasers He/ Cd Krypon 325 nM 355 nM Violet Lasers 405 nM Ar-ion 488 nM He Ne Lasers nM Yellow Dye Lasers 588nM

Absorbtion spectra of some commonly used dyes Wavelength nM Absorbance FITC R-PE APC nM488/565nM 650nM 488 nM 630 nM Excitation wavelength

Dyes used for phenotypic analysis and excitation wavelength 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)* 588nM Rhodamine Texas Red Cascade yellow Cy5.5-APC (APC)* (APC-Cy7)* *Some dyes (……..) can be activated over a broad range of different excitation wavelengths

DNA, RNA and cell viability measurements UV Hoescht (DNA) DAPI (DNA) 488 nM Acridine Orange (DNA) Acridine Orange (RNA) Propidium Iodide (DNA) 7-AAD (DNA) 588nM Ethidium monoazide Pyronin Y

Cell function dyes 633nM SNARF (pH) UV Indo-1 (Ca2+) 488 nM Fluo-3 (Ca2+) Dioxycyanin Rhodamine 123

Emission spectra of commonly used dyes 500nM 600nM 700nM 800nM FITC R-PE ECD APC PE-Cy7 Emission 525nM 578nM 613nM 660nM 760nM

Spectral overlap is detected by different photo-multiplier tubes FL-1 PMT FL-2 PMT FL-4 PMT FL-3 PMT Photo-multiplier tube Electronic compensation FL-1 -% FL-2 FL-2 -% FL-1FL-3 -% FL-2 FL-3 -% FL-4 FL-4 -% FL-3

Electronic compensation FL4-%FL-3 CD56 APC CD3 PerCP No Compensation CD56 APC CD3 PerCP Forward Scatter Side Scatter

Achievable number of parameters: (Modified BD FACStar plus) 1.T- cell differentiation: naïve T-cells Cascade Blue Cascade Yellow FITC PE Cy5PE Cy5.5PE Cy7PE 405nM ExcitationDyeMarker 488nM 595nMAlexa595 APC Cy5.5-APC Cy7-APC CD45RA CD3 CD28 CD49f CD11a CD27 CD4 CD62L CD45RO CD8 CD57 + Forward Scatter + Side Scatter = 13 parameters De Rosa et al, Nature Medicine 2001

Achievable parameters 2. Intracellular signalling Cascade Blue Cascade Yellow ALEXA 546 ALEXA 568 Cy5PE Cy5.5PE Cy7PE 405nM ExcitationDyeMarker 488nM 595nMAlexa595 APC Cy5.5-APC Cy7-APC CD11a CD8 JNK P-AKT-ser473 EMA CD4 CD62L p-TYK2 p-JNK CD28 CD45RA + Forward Scatter + Side Scatter = 13 parameters Perez and Nolan, Nature Biotechnology 2002

References Panchuk-Voloshina N. et al: Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem Sep;47(9): 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 Dec;51(12): 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 Feb;20(2): 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 Feb;7(2): Potential application of multiparameter (polychromatic) flow cytometry. E. mail: