Expanding Multicolor Options in Flow Cytometry with Novel Brilliant Violet TM Fluorophores. Carsten Wiethe Scientific Application Manager BioLegend

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1 Expanding Multicolor Options in Flow Cytometry with Novel Brilliant Violet TM Fluorophores Carsten Wiethe Scientific Application Manager BioLegend

2 Seminar Outline BrilliantViolet TM (BV)fluorophores Instrument configuration and how many colors/marker? Strategiesformulticolorpanel designusing BV TM fluors

3 Types of Fluorochrome Cy5 Alexa 488 Alexa 488 Alexa 488 PE Cy5 Cy5 Synthetic organic dyes: FITC, Alexa Fluor Cy dyes, Dyelight, etc Biological fluorophores: PE APC GFP mcherry Semiconductor nanocrystals: Qdots Crystalplex Organic polymers: Brilliant Violet TM High-Sensitivity Fluorescence

4 Brilliant Violet TM Products Available in 7 formats for the violet laser: -BV421 -BV570 -BV605 -BV650 -BV510 -BV711 -BV785 Extremely bright Out performs alternative fluorochromes for the same detector Stable to fixation, exceptional photostability No special buffers required Effective for surface and intracellular antigens Non-toxic Can be used for sorting and live-cell imaging

5 Brilliant Violet Polymer Family BV fluors are long polymer chains composed of highly efficient, fluorescent units. OR

6 Brightness and Stability of Brilliant Violet Family Members Spleen cells stained with anti-mouse CD4, fixed and analyzed at different times under various conditions

7 Brightness of Brilliant Violet Family Members Fluorochrome Brightness: Stain Index(SI): high background low background Cell count Fluorescence intensity -> degree of spread in the negpopulation

8 Cross-Beam Excitation of Brilliant Violet Family Members BV650 BV570 BV605 BV711 BV785

9 Potential Options for Multicolor Application BV nm BV nm BV nm BV nm BV nm BV nm BV nm

10 Violet Octagon Configuration Standard Standard +1 BV605 BV711 BV711 BV421 BV605 BV510 BV421 BV510 BV650 BV785 BV570 BV650 BV785

11 How many markers to use? 17 color/19 param Typical 405/488/561/ color/17 param Typical 405/488/561/633 OR 405/488/ color/14 param Typical 405/488/561/633 OR 405/488/ color Typical 405/488/633 Canto or Gallios BV421/PacBlue BV510/Zombie Aqua BV570/Zombie Yellow BV605/QD605 BV650/QD655 BV711/QD705 BV785/QD800 BV421/PacBlue BV510/Zombie Aqua BV570/Zombie Yellow BV605/QD605 BV650/QD655 BV711/QD705 BV785/QD800 BV421/PacBlue BV510/Zombie Aqua BV605/QD605 BV650/QD655 BV711/QD705 BV785/QD800 BV421/PacBlue BV510/Zombie Aqua BV605 or BV785 FITC/Alexa488 PerCP(-Cy5.5) PE PE-TR/PE-CF594 PE-Cy5 PE-Cy5.5 PE-Cy7 FITC/Alexa488 PerCP(-Cy5.5) PE PE-TR/PE-CF594 PE-Cy5 PE-Cy7 FITC/Alexa488 PerCP(-Cy5.5) PE PE-TR/PE-CF594 PE-Cy7 FITC/Alexa488 PE PE-TR/PE-CF594 PerCP(-Cy5.5) PE-Cy7 APC/Alexa647 Alexa700 APC-Cy7/Zombie NIR APC/Alexa647 Alexa700 APC-Cy7/Zombie NIR APC/Alexa647 Alexa700 APC-Cy7/Zombie NIR APC/Alexa647 Alexa700 APC-Cy7/Zombie NIR

12 How many markers to use? Try to use as many markers as possible. BUT remember: More Colors= More Power = More Complexity New level of complexity when going beyond 10 colors: - loss of sensitivity - Unwanted FRET - Reagent interactions

13 Developing Multicolor Panels - The Wrong Way FITC BV510 CD4 CD3 BV605 CD25 PE BV650 CD31 CD14 BV421 PE/Cy5 BV785 APC PE/Cy7 BV711 Alexa700 APC/Cy7 CD154 CD39 HLA-DR CD137 CD8 CD45RA CCR7

14 Developing Multicolor Panels - The Better Way Factors to consider: Instrumentconfiguration Brightness of fluorophores expression level, importance & factors affecting expression level (-> include optimization of staining procedure as well!) Spectral spillover, compensation, sensitivity

15 Compensation 3-4 rules to ensure accurate compensation experimental samples and control samples is spectrally matched - identical fluorochrome - identical instrument sensitivity (not PMT-V) background (autofluorescence) is matched for positive and negative control samples positive control is at least as bright as the experimental sample control samples are prepared the same was as experimental samples

16 Spectral Spreading -> Spectral Spreading, Nguyen et al. Cytometry (A), 2013 FITC &PacB/BV421: no loss of sensitivity PerCP/Cy5.5, QD700/BV711, PE/Cy5.5: additive effect may effect sensitivity

17 Spectral Spreading Brilliant Violet Family Members Fluorochromes PerCP- PE- FITC PE Cy5.5 Cy7 APC Alexa700 APC-Cy7 BV421 BV510 BV605 BV650 BV711 BV785 PMT-V FITC PE PerCP- Cy PE/Cy APC Alexa APC/Cy BV BV BV BV BV BV

18 Brilliant Violet

19 Panel Design Laser PMT LP BP Fluorophores 405nm G - 450/50 BV421 F /50 BV510 E /42 BV570 D /12 BV605 C /20 BV650 B /50 BV711 A /60 BV nm C - 488/10 B /30 FITC A /40 PerCP 561nm E /15 PE D /20 PE-Texas Red C /30 PE-Cy5 B /50 PE-Cy5.5 A /60 PE-Cy7 640nm C - 670/14 APC B /45 AlexaFluor700 A /60 APC-Cy7

20 Brilliant Violet : Summary Brilliant Violet Family - 7 members (BV421, BV510, BV570, BV605, BV650, BV711, BV785) =>7 off the violet Bright and stable - Brighter than alternative fluorophores for the same detector - Does not require any special staining buffers - Stable to fixation Expand multicolor applications - More options for violet laser for both surface and intracellular antigens - Can be combined with fixable viabilities dyes (Zombie Aqua, Zombie Yellow, Zombie NIR) Cytometer specifications and instrument setup - Usage depends on violet detector configuration (# of PMT) - Standard filter set - Instrument setup and compensation: easy to manage following basics rules -> Brilliant Violet TM enables > 10 color panels to ALL flow users

21 Thanks! Questions or comments? If you have further questions: Visit us at our Booth (#D012 + E013) Poster: P (Aug25, #351) cwiethe@biolegend.com Please visit our website at: