Imperial College London

Multi-parameter Flow Cytometry: available dyes and combined usage

Martin R. Goodier [email protected]

Department of Immunology 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 Factor production Signal transduction Intracellular calcium pH

Considerations when choosing dye combinations

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

Laser Emission in the UV/ Visible spectrum

700 650 600 Wavelength nM 550 500 450 400 300

RED ORANGE YELLOW GREEN BLUE VIOLET ULTRAVIOLET

He Ne Lasers 633-635 nM Yellow Dye Lasers 588nM Ar-ion 488 nM Violet Lasers 405 nM UV Lasers He/ Cd Krypon 325 nM 355 nM

Absorbtion spectra of some commonly used dyes

Excitation wavelength 488 nM 630 nM FITC R-PE 495nM 488/565nM APC 650nM 400 500 600 Wavelength nM 700

Dyes used for phenotypic analysis and excitation wavelength 405nM 488nM Alexa Fluor 405 Alexa Fluor 430 Cascade Blue (Cascade Yellow)* FITC Alexa Fluor 488 R-PE PE-Texas Red (ECD) PE-Cy5 PerCP PerCP-Cy5.5

PE Cy7 588nM Rhodamine Texas Red Cascade yellow Cy5.5-APC (APC)* (APC-Cy7)* 647 nM APC Alexa Fluor 647 APC-Cy7 *Some dyes (……..) can be activated over a broad range of different excitation wavelengths

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

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

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

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

Electronic compensation

FL4-%FL-3 No Compensation Forward Scatter CD3 PerCP CD3 PerCP

Achievable number of parameters:

(Modified BD FACStar plus) 1. T cell differentiation: naïve T-cells Excitation 405nM Dye Cascade Blue Cascade Yellow 488nM FITC PE Cy5PE Cy5.5PE

Cy7PE Marker CD45RA CD3 CD28 CD49f CD11a CD27 CD4 595nM Alexa595 APC Cy5.5-APC Cy7-APC CD62L CD45RO CD8 CD57 + Forward Scatter + Side Scatter = 13 parameters De Rosa et al, Nature Medicine 2001

Achievable parameters

2. Intracellular signalling Excitation 405nM 488nM Dye Cascade Blue Cascade Yellow ALEXA 546 ALEXA 568 Cy5PE Cy5.5PE

Cy7PE 595nM Alexa595 APC Cy5.5-APC Cy7-APC Marker 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. 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.

Dec;51(12):1699-712. J Histochem Cytochem. 2003

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: [email protected]