1. Multi-parameter Flow Cytometry: available dyes and combined usage Martin R. Goodier m.goodier@ic.ac.uk Department of Immunology Imperial College London Imperial College London

2. 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

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

4. Laser Emission in the UV/ Visible spectrum 700 650 600 550 500 450 400 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 633-635 nM Yellow Dye Lasers 588nM

5. Absorbtion spectra of some commonly used dyes Wavelength nM Absorbance FITC R-PE APC 400 500 600700 495nM488/565nM 650nM 488 nM 630 nM Excitation wavelength

6. 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

7. 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

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

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

10. 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

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

12. 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

13. 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

14. References Panchuk-Voloshina N. et al: Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem. 1999 Sep;47(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 Dec;51(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 Feb;20(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 Feb;7(2):245-8. Potential application of multiparameter (polychromatic) flow cytometry. E. mail: m.goodier@imperial.ac.ukm.goodier@imperial.ac.uk