Lower spectral overlap. Optimized stain index. Innovative antibodies. Superior multicolor flow cytometry. Violet Laser Reagents: The efluor Solution

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1 Lower spectral overlap Optimized stain index Innovative antibodies Superior multicolor flow cytometry Violet Laser Reagents: The efluor Solution

2 ebioscience provides innovative high quality reagents to researchers worldwide that empower the process of scientific discovery in the areas of cellular immunity and oncology. Our extensive portfolio of leading edge cell analysis products and technologies focused on flow cytometry and immunodetection position our customers to be at the forefront of science. We are aligned with, and responsive to, our customers everchanging needs, making ebioscience a preferred discovery partner.

Violet Laser Reagents: The efluor Solution Introduction Flow cytometry is increasingly recognized as a valuable tool for deciphering complex cellular processes and interactions in a variety of

The evolution of flow cytometry as a fundamental tool for the life scientist has been fueled by technological advances in instrumentation that include the introduction of a violet laser as a standard

3 Violet Laser Reagents: The efluor Solution Introduction Flow cytometry is increasingly recognized as a valuable tool for deciphering complex cellular processes and interactions in a variety of systems that model normal and disease states. The evolution of flow cytometry as a fundamental tool for the life scientist has been fueled by technological advances in instrumentation that include the introduction of a violet laser as a standard component of multi-laser flow cytometers. These advances in instrumentation have underscored the need for high quality reagents that allow realization of the power of multicolor flow cytometry. To better support violet laser-based multicolor cytometry, ebioscience has developed a line of efluor reagents compatible with the violet laser (45nm) line. The efluor brand includes both nanocrystal and organic dye-derived fluorescent molecules that have been paired with appropriate antibody specificities to maximize the amount of information that can be acquired from a valuable sample (Figure 1). We currently offer over 1 reagents optimized for use with the violet laser. The growing efluor brand now includes the following fluorophores for use with the violet laser: efluor 45: an organic dye that is an alternative to Pacific Blue in the ebioscience portfolio. efluor 45 emission is slightly red shifted and has a narrower profile compared to Pacific Blue resulting in less overall compensation. efluor 65 Nanocrystals: a brighter alternative to Pacific Orange that features very narrow emission spectra to maximize signal to noise and minimize compensation. efluor 65 Nanocrystals: our newest and brightest nanocrystal represents a third fluor for the violet laser. In contrast to AmCyan, efluor 65 NC features a very narrow emission profile that maximizes signal to noise and minimizes compensation requirements on the violet laser. efluor Reagents for the Violet Laser Figure 1: efluor reagents for the violet laser. To illustrate the quality performance of efluor reagents for the violet laser, staining examples for % of Max efluor CD45RA (clone HI1) 1 efluor 45 8 Pacific Blue % of Max efluor 65 NC CD8 (clone ) 1 efluor 65 NC 8 Pacific Orange % of Max efluor 65 NC B22 (clone RA3-6B2) efluor 65 NC Qdot 655 each fluorophore are shown. Human peripheral blood cells were stained with efluor 45 anti-human CD45RA or the same antibody clone conjugated to Pacific Blue (left plot). Mouse splenocytes were stained with efluor 65 NC anti-mouse CD8 or the same clone conjugated to Pacific Orange (middle plot) and efluor 65 NC anti-mouse B22 or the same clone conjugated to Qdot 655 (right plot).

4 The efluor Solution for the Violet Laser The efluor solution for the violet laser provides three fluorophores that work together to provide maximum resolution of antigenic determinants when used simultaneously. This is accomplished by narrow emission peaks with little spectral overlap and is in stark contrast to the broad emission peaks and spectral overlap seen with the traditional approach for the violet laser (Figure 2). Our broad portfolio of biological content permits ideal pairing of specificity with fluorophore that allows straightforward solutions for the violet laser. Figure 2: Emission spectra of traditional and efluor reagents for the violet laser. Normalized emission spectra of the traditional fluorophores commonly used with the violet laser (top) versus the efluor solution (bottom). The advantageous narrow emission spectra of the efluor solution is evident when compared to the broad emission spectra and large spectral overlap of the traditional approach. The recommended collection filters for each fluorophore are indicated. Emission Spectra of Traditional and efluor Reagents Traditional Approach Fluoresence (arbitrary units) /5 525/5 585/ Emission Wavelength (nm) Pacific Blue AmCyan Pacific Orange efluor Solution Fluoresence (arbitrary units) /5 65/4 66/ Emission Wavelength (nm) efluor 45 efluor 65 NC efluor 65 NC The efluor Nanocrystals Advantage The narrow emission profiles of the efluor solution are derived from the properties of efluor Nanocrystals. The semiconductor material-based particles are approximately protein sized (2-1nm) and behave as a fluorophore in biological applications. Their unique spectral properties include preferential excitation at shorter wavelengths (Figure 3) and a tendency towards extremely long stoke shifts. Figure 3: Absorption and emission spectra. The absorption (dashed line) and emission (solid line) spectra of the range of efluor Nanocrystals. Absorption and Emission Spectra of efluor Nanocrystals Fluoresence (arbitrary units) Emission Wavelength (nm) efluor 525 NC efluor 565 NC efluor 65 NC efluor 625 NC efluor 65 NC

5 ebioscience efluor Nanocrystals are constructed in a three step process (Figure 4). First, the wavelength specific core particle is synthesized and then sealed and protected with a Zinc-Sulfide shell (Step A). The second step involves the encapsulation of nanocrystals in a proprietary lipid coating (Step B) that ensures low background staining and also provides a stable platform for the final step of covalent coupling of antibodies to create each unique reagent (Step C). efluor Nanocrystals Composition Figure 4: efluor Nanocrystals composition. An illustration of the layered composition of efluor Nanocrystals particles designed for life science applications. Step A Step B Step C Only efluor Nanocrystals feature a unique proprietary coating that confers a combination of low fluorescence background and substantial elimination of aggregate phenomena often associated with other nanocrystal preparations. Designing an Effective Multicolor Solution An effective multicolor reagent panel is one that is robust in use and provides effective resolution for reproducible and accurate biological assessment. The use of the so called Stain Index, or SI, is an objective measure of how well resolved a given cell population is when analyzed using a particular instrument with its unique configuration and settings. Figure 5 graphically illustrates the SI calculation (left) and the actual SI values for ebioscience anti-mouse CD4 reagents (right). The SI is calculated as the difference in the means of the positive and negative populations (D) divided by two times the standard deviation of the negative peak (W). As can be seen in Figure 5, the applied compensation will increase the fluorescence spread of the negative population and subsequently decrease the resolution of the positive population from the negative. Stain Index Figure 5: Stain Index - a measure of staining resolution. A graphical representation of the stain index 7 6 W unstained Stain Index (SI) = D / W APC PE PE-Cy7 Alexa Fluor calculation is shown on the left. The graphic on the right represents the calculated stain index for anti-mouse CD4 conjugated to the range of ebioscience fluorophores and used to stain mouse splenocytes D W compensated dye efluor 65 NC PerCP-Cy5.5 Alexa Fluor 647 APC-eFluor 78 efluor 45 efluor 65 NC FITC Alexa Fluor

6 efluor Provides a Better Relative Stain Index A simple comparison experiment was performed to assess the efficacy of the efluor solution vs. the traditional approach. Human PBMC were stained for CD3, CD4, and CD8 using antibodies directly conjugated to traditional fluorophores (traditional approach) or efluor fluorophores (efluor solution). Samples were stained according to manufacturers recommended conditions, and acquired on a violet laser (5mW) equipped flow cytometer (BD LSRII), with compensation and filter sets optimized for each set of fluorophores. The resulting data plots and compensation values are shown in Figure 6. Using the traditional approach, considerable amounts of spectral compensation were required in order to acquire usable data. Data from cells stained using the traditional approach is shown below (Figure 6A). A consequence of the compensation requirement is that significant data spread in the CD4 population and a decrease in CD8 resolution are observed that result from spillover of the AmCyan signal into the detectors for Pacific Blue and Pacific Orange respectively. The cumulative effect is a greatly reduced overall stain index (Figure 7). In contrast, cells stained using the efluor solution (Figure 6B) require minimal compensation and the data clearly shows brighter CD8 staining with resolution of CD8 dim/nk cells, an absence of data spread, and the ability to unambiguously identify the CD4/CD8 double positive T cells that are not resolved when traditional approaches are employed. Figure 6: Comparison of the traditional approach with the efluor solution. Human PBMC were stained with the traditional fluorophores AmCyan anti-human CD3, Pacific Blue anti-human CD4, and Pacific Orange anti-human CD8 (A) or with the efluor solution of efluor 65 NC anti-human CD3, efluor 45 anti-human CD4, and efluor 65 NC anti-human CD8 (B). The required compensation values are shown in table form to the left of the data plots. Comparison of the Traditional Approach with the efluor Solution A: Traditional Approach Minus % Fluorochrome in Detector Stain Pacific Blue AmCyan Pacific Orange CD4 Pacific Blue CD3 AmCyan % of Max CD8 Pacific Orange CD8 Pacific Orange CD3 AmCyan CD4 Pacific Blue B: efluor Solution 1 Minus % Fluorochrome in Detector Stain efluor 45 efluor 65 NC efluor 65 NC CD4 efluor CD8 efluor 65 NC % of Max CD8 efluor 65 NC CD3 efluor 65 NC CD3 efluor 65 NC CD4 efluor 45

7 In order to objectively measure the visually superior performance of the efluor solution in this multicolor example, stain indexes were calculated to quantitatively assess the benefits of the efluor approach (Figure 7). The efluor solution offered improved resolution for each of the three specificities evaluated for detection with the violet laser. The most significant advantage in this panel was the improved resolution of the CD8 population. The ongoing expansion of the efluor product line from ebioscience brings researchers the needed tools to capitalize on their instrument investment by providing reagents to take advantage of the full capacity of their cytometer. Relative Stain Index Specificity Fluor Relative Stain Index % efluor Advantage CD3 efluor 65 NC 34 AmCyan 3 13 % CD4 efluor Pacific Blue % CD8 efluor 65 NC 51 Pacific Orange % Figure 7: Relative stain index of the efluor solution versus traditional fluorophores. From the data shown in Figure 6, the relative stain index was calculated from the compensated data for each antibody used in the staining panels. The percent improvement offered by the efluor reagents over the traditional fluorophores is shown for each specificity. Conclusion: efluor Solution - Tailored for the Violet Laser The development of flow cytometers equipped with a violet laser has helped drive recognition of multiparameter flow cytometry as a must have tool for the life sciences. Realizing the potential of the violet laser requires robust and reliable reagents as well as relevant specificities for easy integration into any multicolor staining panel. A primary goal of ebioscience is to combine our industry leading expertise with our innovative spirit to develop optimized reagents for multicolor flow cytometry. In order to validate performance of the efluor solution for the violet laser, a three color staining panel consisting of CD3 efluor 45, CD45RA efluor 65 NC, and CD45RO efluor 65 NC was used to discriminate human naïve and memory T cells. As shown in Figure 8, this panel easily resolved the CD3-expressing cells and showed the expected distribution of CD45RA and CD45RO. The efluor solution is an optimized fluorophore solution designed to maximize the power of your violet laserequipped cytometer. By combining new organic dyes, proprietary biocompatible nanocrystal technologies and an extensive antibody portfolio, ebioscience has developed a uniquely powerful fluorescent reagent system for the violet laser. Naïve and Memory T Cell Discrimination Using the efluor Solution # of Cells CD45RO efluor 65 NC Figure 8: Naïve and memory T cell discrimination using the efluor solution. Human PBMC were stained with efluor 45 anti-human CD3, efluor 65 NC anti-human CD45RA, and efluor 65 NC anti-human CD45RO to visualize naïve and memory CD3+ T cells CD3 efluor 45 NC CD45RA efluor 65 NC

8 NOW AVAILABLE: Products for the Violet Laser All Products are for research use only. Human Description Clone Isotype efluor 45 Pacific Blue efluor 65 NC efluor 65 NC CD1a HI149 migg CD3 OKT3 migg2a UCHT1 migg CD4 OKT4 migg2b RPA-T4 migg CD8α OKT8 migg2a CD8α RPA-T8 migg CD11b ICRF44 migg CD14 61D3 migg CD15 HI98 migm CD16 CB16 migg CD19 HIB19 migg CD2 2H7 migg2b CD27 O323 migg CD44 IM7 rigg2b CD45 (LCA) HI3 migg CD45RA HI1 migg2b CD45RO UCHL1 migg2a CD62L DREG-56 migg CD95 DX2 migg CD117 YB5.B8 migg CD123 6H6 migg CD127 RDR5 migg CD282 T2.5 migg CD284 HTA125 migg2a Foxp3 236A/E7 migg Foxp3 PCH11 rigg2a HLA-DR LN3 migg2b IFNγ 4S.B3 migg IL-1 JES3-9D7 rigg IL-12/IL-23 p4/7 C8.6 migg TNFα MAb11 migg Please visit ebioscience.com as new violet laser products are launched regularly. Mouse Description Clone Isotype efluor 45 Pacific Blue efluor 65 NC efluor 65 NC CD3 17A2 rigg2b CD3 5A2 hamigg CD4 GK1.5 rigg2b CD4 RM4-5 rigg2a CD8α rigg2a CD11b M1/7 rigg2b CD11c N418 hamigg CD16/32 93 rigg2a CD19 1D3 rigg2a CD21/CD35 4E3 rigg2a CD24 M1/69 rigg2b CD31 39 rigg2a CD34 RAM34 rigg2a CD44 IM7 rigg2b CD45 (LCA) 3-F11 rigg2b

9 Description Clone Isotype efluor 45 Pacific Blue efluor 65 NC efluor 65 NC CD45R (B22) RA3-6B2 rigg2a CD45.1 A2 migg2a CD62L MEL-14 rigg2a CD69 H1.2F3 hamigg CD73 TY/11.8 rigg CD86 GL1 rigg2a CD9.1 HIS51 migg2a CD rigg2a CD12 3C4 rigg2a CD15 MJ7/18 rigg2a CD122 TM-b1 rigg2b CD127 A7R34 rigg2a CD282 T2.5 migg CD39 Avas12a1 rigg2a F4/8 BM8 rigg2a Foxp3 FJK-16s rigg2a IFNγ XMG1.2 rigg IgD 11-26c rigg2a IgM II/41 rigg2a IL-2 JES6-5H4 rigg2b Ly-6A/E D7 rigg2a Ly-6G RB6-8C5 rigg2b MHC Class II (I-A/I-E) M5/ rigg2b NK-1.1 PK136 migg2a TER-119 TER-119 rigg2b TNFα MP6-XT22 rigg Mouse Hematopoietic Lineage Flow Cocktail Various Controls Description Isotype efluor 45 Pacific Blue efluor 65 NC efluor 65 NC IgG Isotype Control hamigg IgG Isotype Control hamigg IgG1,k Isotype Control migg IgG2a,k Isotype Control migg2a IgG2b Isotype Control migg2b IgM Isotype Control migm IgG1 Isotype Control rigg IgG2a Isotype Control rigg2a IgG2b Isotype Control rigg2b IgG2b Isotype Control rigg2b IgM Isotype Control rigm Streptavidin NA Support Products Description Catalog No. Description Catalog No. Red Blood Cell Lysis Buffer, 1X Brefeldin A Solution -456 Flow Cytometry Staining Buffer Monensin Solution -455 efluor NC Flow Cytometry Staining Buffer Foxp3 Fixation/Permeabilization Concentrate Human FcγR-Binding Inhibitor Foxp3 Fixation/Permeabilization Diluent Anti-Mouse CD16/32 - Blocks Fc Binding Foxp3 Fixation/Permeabilization Concentrate and Diluent Normal Mouse Serum Foxp3 Staining Buffer Set Normal Rat Serum Propidium Iodide Staining Solution -699 IC Fixation Buffer AAD Viability Staining Solution Please visit ebioscience.com as new violet laser products are launched regularly.

10 NOTES:

11 INSTRUMENT & FLUOROCHROME: Compatibility Chart INSTRUMENTS Qdot is a registered trademark of Quantum Dot Corporation. Alexa Fluor and Texas Red are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. Pacific Blue and Pacific Orange are trademarks of Molecular Probes, Inc. BD FACSAria, BD FACSCalibur, and BD FACSCanto are trademarks of Becton, Dickinson and Company. FC5 is a trademark of Beckman Coulter, Inc. CYAN and MoFlo are registered trademarks of Dako Denmarks A/S. Accuri C6 is a registered trademark of Accuri Cytometers, Inc. Quanta is a registered trademark of NPE Systems, Inc. CyFlow is a registered trademark of CyTecs GmbH. Reflection is a registered trademark of icyt Mission Technology, Inc. efluor is a trademark of ebioscience, Inc. i-cyt Reflection LSR II BD FACSAria BD FACSCanto II BD FACSCalibur CYAN FC5 MoFloXDP Accuri C6 JSAN APC-eFluor 78 (78 nm) APC-Alexa Fluor 75 (775 nm) 633 nm Excitation Laser APC-Cy7 (76 nm) Alexa Fluor 7 (723 nm) FLUOROCHROMES (peak emission) 488 nm Excitation Laser 45 nm Excitation Laser Alexa Fluor 647 (668 nm) APC (66 nm) PE-Cy7 (785 nm) PE-Cy5.5 (695 nm) PerCP-Cy5.5 (69 nm) PerCP (675 nm) PE-Cy5 (667 nm) PE-Texas Red (615 nm) PE (575 nm) Alexa Fluor 488 (519 nm) FITC (518 nm) CFSE (517 nm) GFP (59 nm) efluor 65 NC (65 nm) efluor 625 NC (625 nm) efluor 65 NC (65 nm) Pacific Orange (551 nm) AmCyan (491 nm) Pacific Blue (455 nm) efluor 45 (45 nm)

12 North America ebioscience, Inc Science Center Drive, San Diego, CA 92121, USA Tel: Fax: Customer Service: Tel: Technical Support: Tel: United Kingdom ebioscience, Ltd. 2nd Floor, Titan Court 3 Bishop Square Hatfield, AL1 9NA Tel: +44 () Fax: +44 () Customer Service: Tel: +44 () uk@ebioscience.com Technical Support: Tel: +44 ()

efluor Organic Dyes 450/50 BP Fluorescence Intensity Wavelength (nm)

efluor Organic Dyes 450/50 BP Fluorescence Intensity Wavelength (nm) efluor Organic Dyes efluor Organic Dyes Catalog No. Description Clone Application Anti-Mouse efluor 450 Products 48-0042 Anti-mouse CD4 RM4-5 Flow Cytometry 48-0081 Anti-mouse CD8 53-6.7 Flow Cytometry