Complete Brilliance. Brilliant Violet. Novel Fluorophore Conjugates for Flow Cytometry. for the Violet Laser. Antibody Conjugates

Brilliant Violet Antibody Conjugates Complete Brilliance for the Violet Laser Novel Fluorophore Conjugates for Flow Cytometry Toll-Free Tel: (US & Canada): 1.877.BIOLEGEND (246.5343) Tel: 858.768.58 biolegend.com 2-5-1 World-Class Quality Superior Customer Support Outstanding Value

Introduction Brilliant Violet antibody conjugates, proudly co-developed by BioLegend and Sirigen, are an innovative class of novel research reagents, providing more options for multicolor flow cytometry panels and superior results. Brilliant Violet Technology The Brilliant Violet family of fluorescent molecules are organic polymers with an extraordinary capacity to absorb energy (extinction coefficient) and a high efficiency with which to convert that absorbed energy to an emitted signal (quantum yield). When conjugated to antibodies, this results in high intensity brightness on labeled cells. Physical Properties Brilliant Violet 421 has an extinction coefficient of 2,5, M -1 cm -1 at 45 nm, an aqueous solution quantum yield of 65 ± 5%, and solubility in excess of 5 mg/ml in PBS. The extinction coefficient contributes to its superior brightness compared to Pacific Blue, which has an extinction coefficient of 3, M -1 cm -1. Brilliant Violet 421 can also be modified with another dye to produce high stokes shift emissions. Brilliant Violet 57, Brilliant Violet 65, Brilliant Violet 65, Brilliant Violet 711, and Brilliant Violet 785 are such derivatives of the Brilliant Violet 421 polymer, emitting maximally at 57 nm, 63 nm, 645 nm, 711 nm, and 785 nm, respectively, when excited at 45 nm. Brilliant Violet 51, another High Sensitivity Fluorescence polymer, is the newest violet laser excitable fluorophore with maximal emission at 51 nm. This figure provides the emission spectra of the Brilliant Violet fluorophores. To view excitation data and to compare with other fluorophores, use our Spectra Analyzer tool at: biolegend.com/spectraanalyzer. Emission Spectra of Brilliant Violet Fluorophores Easy to Use and Trouble-Free Brilliant Violet antibody conjugates are simple to use, compatible with standard staining buffers, and stable to fixation. Provided in convenient 5 µl test sizes at optimal ready-to-use concentrations, our Brilliant Violet antibody products can easily be added to your multicolor panels. We are now introducing µg sizes for optimal value. For more information, visit: biolegend.com/brilliantviolet 2 Customer Service: 858-768-58

Brilliant Violet Fluorophore Family at a Glance Brilliant Violet 421 Brilliant Violet 51 Brilliant Violet 57 Excitation max = 45 nm Excitation max = 45 nm Excitation max = 45 nm Emission max = 421 nm Emission max = 51 nm Emission max = 57 nm Recommended filter = 45/5 Recommended filter = 51/5 Recommended filter = 585/42 Comparable Fluorophores Comparable Fluorophores Comparable Fluorophores Pacific Blue BD Horizon V45 BD Horizon V5 Pacific Orange Qdot 565 Alexa Fluor 45 Cascade Blue AmCyan Cascade Yellow efluor 565NC efluor 45 Qdot 545 Krome Orange Brightness = 5 On a scale from 1 to 5, with 5 being the brightest. Molar Extinction Coeff.= 2,5, M -1 cm -1 Quantum Yield =.65 in DPBS Brightness = 3 Molar Extinction Coeff.= 577, M -1 cm -1 Quantum Yield =.44 in DPBS MW = 6-8 kd MW = 6-8 kd MW = 6-8 kd Brightness = 2-3 Molar Extinction Coeff.= 2,3, M -1 cm -1 Quantum Yield =.8 in DPBS Brilliant Violet 65 Brilliant Violet 65 Brilliant Violet 711 Excitation max = 45 nm Excitation max = 45 nm Excitation max = 45 nm Emission max = 63 nm Emission max = 645 nm Emission max = 711 nm Recommended filter = 61/2 Recommended filter = 66/2 Recommended filter = 71/5 Comparable Fluorophores Qdot 65 efluor 65NC Brightness = 4 Molar Extinction Coeff.= 2,4, M -1 cm -1 Quantum Yield =.29 in DPBS Comparable Fluorophores Qdot 655 efluor 65NC Brightness = 3 Molar Extinction Coeff.= 2,5, M -1 cm -1 Quantum Yield =.17 in DPBS Comparable Fluorophores efluor 7NC Qdot 75 MW = 6-8 kd MW = 6-8 kd MW = 6-8 kd Brilliant Violet 785 Excitation max = 45 nm Emission max = 785 nm Recommended filter = 78/6 Comparable Fluorophores Qdot 8 Brightness = 3 Molar Extinction Coeff.= 2,8, M -1 cm -1 Quantum Yield =.15 in DPBS Brightness = 3 Molar Extinction Coeff.= 2,5, M -1 cm -1 Quantum Yield =.4 in DPBS MW = 6-8 kd 3 biolegend.com

Brilliant Violet Considerations for Multicolor Flow Cytometry Brilliant Violet 421 (BV421 ) BV421 has an emission spectrum that is more narrow than that of Pacific Blue ; thus, there is less spillover into neighboring channels, such as AmCyan or Horizon V5. BV421 is excitable to some degree by the UV laser (35-355 nm) (~25% of maximal excitation), so some compensation is required when used in combination with UV-excited fluorophores. However, BV421 is consistently demonstrated to be compatible with viability probes Fixable Live/Dead Blue or DAPI excited off the UV laser. BV421 is not excited by other lasers, such as the 488, 532 or 561 nm. For most cell surface staining, BV421 is significantly brighter than equivalent fluorophores, including Pacific Blue, BD Horizon V45, efluor 45, and Alexa Fluor 45. It has an extinction coefficient of 2,5, M -1 cm -1 at 45 nm, and an aqueous solution quantum yield of 65 ± 5%. On a brightness scale of 1-5, with 5 being the brightest, we give this a 5. It may be possible to use BV421 together with Pacific Blue, but very specific and narrow bandpass filters would be needed. BioLegend does not recommend attempting this for the average flow user, as compensation requirements will likely be very high. In addition, stray light from the violet laser can increase the background signal for BV421. Brilliant Violet 51 (BV51 ) BV51 has a similar emission spectrum to that of Horizon V5, so we recommend using the bandpass filter setting being used for Horizon V5 or Live/Dead Aqua (51/5 or 525/5). BV51 is excitable by the UV laser (35-355 nm), so some compensation is required when used in combination with UV-excited fluorophores. BV51 is not excited by other lasers, such as the 488, 532 or 561 nm. For cell surface staining, BV51 is significantly brighter than equivalent fluorophores, including Pacific Orange and BD Horizon V5. On a brightness scale of 1-5, with 5 being the brightest, we give this a 3. When using BV51 with other Brilliant Violet fluorophores, compensation and PMT voltage balancing with BV57 and some BV65 is required. A good strategy when choosing markers for BV51 and BV57 is to choose markers that will not be co-expressed on the same cell. 4 Customer Service: 858-768-58

Brilliant Violet 57 (BV57 ) BV57 is excited by the violet laser at 45 nm, emits maximally at 57 nm, and can be used in place of Pacific Orange, Qdot 565, Qdot 585, efluor 565NC, and efluor 585NC. BV57 antibody conjugates provide a good signal-to-noise ratio, although not as bright as BV421. On a brightness scale of 1-5, with 5 being the brightest, we give this a 2-3. The optimal bandpass filter (585/42) is typically not the default filter on most instruments. Be sure that this bandpass filter is correctly configured on the instrument before using BV57. Use the 575LP filter when using BV57 with Horizon V5 or Fixable Live/Dead Aqua, in order to prevent unnecessary spillover and with instruments equipped with 561 nm laser line. In most other cases, using a LP filter between 545 and 556 nm is acceptable. BV57 has an emission spectrum very similar to that of PE and it can be partially excited by the green laser (532 nm), the yellow-green laser (561 nm), and to a lower extent, by the blue laser (488 nm). This raises potential compensation issues when using the two fluorophores together in a multicolor panel. In order to minimize spill-over/compensation requirements for the PE channel, we advise that users adjust the PMT-V for BV57 to be higher than PMT-V for PE. The data below is an example of % compensation requirements for two PMT-voltage scenarios, one in which the BV57 PMT-V is higher and one where the PE PMT-V is higher. Note that the % compensation into BV421 and PE is significantly less when the BV57 PMT-V is higher. PMT BV57 Voltage PE 5.7% 52 BV421 6.% 536 BV57 55 PE 2.42% 6 BV421 12.57% 6 BV57 55 *This is only an example for one specific instrument and configuration. Optimization will be required for your specific instrument and configuration. Using a 575LP for the PE detector also helps to reduce the spillover of BV57 into PE. Brilliant Violet 65 (BV65 ) BV65 is excited by the violet laser at 45 nm and emits maximally at 63 nm, and can be used in place of Qdot 65 and efluor 65NC. BV65 antibody conjugates provide excellent signal-to-noise ratio with brightness on par with that of BV421 or PE. On a brightness scale of 1-5, with 5 being the brightest, we give this a 4. BV65 requires very little compensation with other lasers. When used in a panel with BV57 and BV65 on the violet laser, PMT voltage balancing is required, which means that the default CS&T settings will likely not be optimal when using these together. The standard Qdot 65 filter for this PMT, 61/2 with a 595LP dichroic, works well for BV65 detection. 5 biolegend.com

Brilliant Violet Considerations for Multicolor Flow Cytometry (Continued) Brilliant Violet 65 (BV65 ) BV65 is excited by the violet laser at 45 nm and emits maximally at 645 nm, and can be used in place of Qdot 655 and efluor 65NC. BV65 antibody conjugates provide excellent signal-to-noise ratio, with brightness rated at 3 on a scale of 1-5, with 5 being the brightest. BV65 has some slight compensation requirements with APC, due to its partial excitation by the 633 nm laser. The compensation requirements are minimal and do not normally require any special adjustments. The standard Qdot 655 filter for this PMT, 66/2 with a 63nm LP dichroic, works well for BV65 detection. When being used together with BV65, we recommend the 66/2 or 67/2 BP filter to minimize spillover. Brilliant Violet 711 (BV711 ) BV711 is excited by the violet laser at 45 nm and emits maximally at 711 nm, and can be used in place of Qdot 75 and efluor 7NC. BV711 antibody conjugates provide good brightness, rated at 3 on a scale of 1-5, with 5 being the brightest. The standard Qdot 7 filter for this PMT, 71/5 with a 685nm LP dichroic, works well for BV711 detection. BV711 has some spillover into 633 nm laserexcited fluorophores, due to its partial excitation by the 633 nm laser, as well as moderate spillover into PerCP/Cy5.5. The compensation requirements are manageable and do not normally require any special adjustments. BV711 will have moderate spillover into BV785. Compensation requirements may be high, but manageable. When used together, we recommend BV785 for more highly expressed antigens and BV711 for lower expressing antigens. 6 Customer Service: 858-768-58

Brilliant Violet 785 (BV785 ) BV785 is excited by the violet laser at 45 nm, emits maximally at 785 nm, and can be used in place of Qdot 8. BV785 antibody conjugates provide good brightness, rated at 3 on a scale of 1-5, with 5 being the brightest. BV785 has some cross-beam excitation by the red laser and thus a small amount of spillover into the APC/Cy7 channel. The standard Qdot 8 filter for this PMT, 78/6 with a 75 nm LP dichroic, works well for BV785 detection. Brilliant Violet 421 in Immunofluorescence Microscopy BL/6 mouse lymph nodes, stained with CD19-BV421 (red) and CD4-Alexa Fluor 488 (green). Image provided by Ann Haberman and Christine Podolski, Yale University. BL/6 mouse lymph nodes, stained with I-A/I-E BV421 (red), B22 Alexa Fluor 488 (blue), and CD11c FITC (green). Image provided by Ann Haberman and Christine Podolski, Yale University. 7 biolegend.com

Brilliant Results Brilliant Violet antibody conjugates have been thoroughly tested by our in-house scientific team, as well as by external testers, to ensure that our products are at the highest level of quality. Products have been validated by various labs worldwide, on a variety of instruments, using a wide variety of applications, including flow cytometry and confocal microscopy. Testing not only ensures the usefulness of Brilliant Violet products in these applications, but also verifies that the products are easy-to-use, simple to integrate into complex multicolor panels, and effective at producing the best possible results. Brilliant Violet 421 High sensitivity for mouse CD127 Cat. No. Description Clone 13523/4 Brilliant Violet 421 anti-mouse CD127 (IL-7Rα) A7R34 Brilliant Sensitivity The high intensity brightness of Brilliant Violet 421 enables visualization of weakly expressed antigens. It can also allow researchers to use higher dilutions of antibody to obtain the same data, resulting in less antibody per test. Here, researchers were able to use dilutions of Brilliant Violet 421 anti-mouse CD127 to identify precursor cells from bone marrow in a 6-color panel. At all dilutions tested, the BV421 outperformed the Pacific Blue equivalent used at much higher concentrations (1 µg/ml). The BV421 results were comparable to those of the PE conjugate. The total amount of antibody was critical in this sorting experiment because the desired population was less than 2% of total stained cells, and the starting amount was 5 million bone marrow cells. 8 Customer Service: 858-768-58

Data Courtesy of Renee De Pooter and David Leclerc, University of Chicago..5 µg/ml.25 µg/ml.13 µg/ml.6 µg/ml 1 5 1 5 1 5 1 5 APC-CD117 1 4 1 3 1 2 1 4 1 3 1 2 1 4 1 3 1 2 1 4 1 3 1 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 BV421 -CD127 APC-CD117 1 5 1 4 1 3 1 2 1 5 1 4 1 3 1 2 1 2 1 3 1 4 1 5 PE-CD127 1 2 1 3 1 4 1 5 PB-CD127 9 biolegend.com

Brilliant Violet 421 Resolution of mouse NK1.1 populations Cat. No. Description Clone 18731/2 Brilliant Violet 421 anti-mouse NK-1.1 PK136 Brilliant Resolution Resolution is critical for identifying both rare cell populations and dimly expressed markers. NK1.1 is commonly used for studying NK and NKT cells in the mouse model. Here, researchers demonstrate the resolving power of Brilliant Violet 421, with brightness on par with PE-conjugated antibody, resulting in similar population percentages. The BV421 antibody is able to clearly resolve the NK and NKT cell populations, providing far superior signal-to-noise compared to BD Horizon V45 antibody. Resolution of human CD45RA vs. CD45RO populations Cat. No. Description Clone 34129/3 Brilliant Violet 421 anti-human CD45RA HI1 4341/2 Brilliant Violet 421 Mouse IgG2b, κ Isotype Ctrl MPC-11 Brilliant Resolution Resolution is critical for identifying both transitioning cell populations and fully differentiated populations. In T-cell studies, CD45RA is used to identify naïve T cells, while CD45RO identifies activated and memory T cell populations. Here, researchers demonstrate that Brilliant Violet 421 -conjugated anti-cd45ra gives a significantly improved (one log greater) dynamic range compared to efluor 65NC-conjugated antibody (A and B). The BV421 -antibody conjugate allows for visualization of transitioning populations and clearly distinguishes the fully differentiated memory population from the naïve population. The isotype control at the same concentration provides a very low background, further demonstrating the increased dynamic range of BV421 antibodies, with highly bright positive populations and low non-specific staining. 1 Customer Service: 858-768-58

Data courtesy of Eva Tolosa and Timo Lischke at UKE Hamburg. 1 5 4.3% 1 5 4.8% 1 5 4.4% CD3-PerCP 1 4 1 3 CD3-PerCP 1 4 1 3 CD3-PerCP 1 4 1 3-58 -479-435 -12 1 2 1 3 1 4 1 5 NK1.1-BV421 NK1.1-V45 NK1.1-PE -877-12 1 2 1 3 1 4 1 5 6-378 -12 1 2 1 3 1 4 1 5 Data Courtesy of Marcin Pekalski and Linda Wicker at Cambridge Institute for Medical Research, Diabetes and Inflammation Lab. 1 5 1 5 1 5 1 4 1 4 1 4 FITC 1 3 FITC 1 3 PE 1 3 1 2 1 2 1 2 1 2 1 3 1 4 1 5 CD45RA-BV421 1 2 1 3 1 4 1 5 CD45RA-eFluor65 1 2 1 3 1 4 1 5 CD45RA-Brilliant Violet 421 11 biolegend.com

Brilliant Violet 421 Tetramer Resolution mouse CD1d Cat. No. Description 45225/6 Streptavidin-Brilliant Violet 421 Brilliant Sensitivity The high intensity brightness of Brilliant Violet 421 enables high resolution of rare tetramer-stained populations. Tetramer assays are designed to identify antigen-specific T cell populations through the use of peptide-loaded MHC tetramers labeled with a fluorochrome. Here, researchers compared PBS57-loaded mouse CD1d tetramers labeled with Streptavidin-Brilliant Violet 421, PE, or Pacific Blue. BV421 outperformed both PE and Pacific Blue at equivalent tetramer concentrations. In this scenario, the Pacific Blue signal is too weak to provide any resolution of positive populations. BV421 provided excellent signal-to-noise at all concentrations tested. Functional cells post-sorting with mouse CD4 Cat. No. Description Clone 1437/8 Brilliant Violet 421 anti-mouse CD4 GK1.5 Brilliant Viability Cell viability is critical after cell sorting. Here, pooled Balb/c spleen and lymph node cells were stained with CD4-BV421 or CD4-Pacific Blue and sorted for CD4 positive cells. The cells were then labeled with CFSE and stimulated in 96-well flat-bottom plates coated with anti-cd3/anti-cd28 (1 µg/ml each) at 1 x 1 5 cells/well. On day four, cells were analyzed for CFSE dilution. Histograms A and B demonstrate comparable division and expansion of cells sorted with BV421 compared to those sorted with Pacific Blue, as indicated by the loss of CFSE signal. Unstimulated cells are displayed as the gray histogram. Figure C shows the median fluorescence values of each group. Error bars represent the standard deviation of triplicate wells. Figure D demonstrates that the % of cells that have divided is comparable between BV421 and Pacific Blue -stained cells. Overall, the data shows excellent viability and responsiveness of cells after staining and sorting with a BV421 antibody. 12 Customer Service: 858-768-58

Data Courtesy of Rick Willis and John Altman, NIH Tetramer Core Facility at Emory/Yerkes. 1:4 1:8 1:16 1:32 mcd1d PBS57 tetramer PE BV421 Pacific Blue CD3 FITC Data provided by Aras Toker and Jochen Hühn, Helmholtz Centre for Infection Research. A. BV421 CD4-sorted B. PB CD4-sorted 1 1 8 8 % of Max 6 % of Max 6 4 4 2 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 CFSE-A CFSE-A C. D. 1 7 GMFI (CFSE) 5 5 4 3 2 1 % divided 95 9 1 5 BV PacBlue unstim BV PacBlue unstim 13 biolegend.com

Brilliant Violet 421 Resolution of human CD56 populations Cat. No. Description Clone 318327/8 Brilliant Violet 421 anti-human CD56 (NCAM) HCD56 Brilliant Resolution Resolution is critical for identifying both rare cell populations and dimly expressed markers. For human NK cells, the labeling of CD56 is commonly used for flow cytometry to identify CD56-negative, CD56-positive and CD56-bright populations. Here, researchers demonstrate the resolving power of Brilliant Violet 421, with brightness on par with PE/Cy7-conjugated antibody, resulting in similar population percentages. The BV421 antibody is able to clearly resolve the NK cell populations, providing far superior signal-to-noise compared to Pacific Blue antibody. Multicolor Microscopy with human CD56 Cat. No. Description Clone 318327/8 Brilliant Violet 421 anti-human CD56 (NCAM) HCD56 Brilliant Imaging The high intensity brightness and exceptional photostability of Brilliant Violet 421 enable for the first time, visualization of antigens using directly-labeled antibody conjugates in the blueemitting channel for microscopy. Here, researchers stained NK92 cells (human NK cell line, top row) or NK92 cells mixed with K562 cells (bottom row) with anti-cd56 BV421, anti-perforin FITC, and phalloidin Alexa Fluor 568, and acquired images on an Olympus IX81 spinning disk confocal microscope on 1X objective, NA 1.45. Exposures: 488 = 1 ms, 568 = 1 ms, BV421 (45 nm) = 2 ms. CD56-expressing NK cells were easily distinguished from the CD56-non-expressing K562 cells. 14 Customer Service: 858-768-58

Data courtesy of Axel Schulz and Andreas Thiel at BCRT/Charite Berlin. 1 5 1 5 1 5 1 4 1 4 1 4 SSC-A 1 3 SSC-A 1 3 SSC-A 1 3 1 2 1 2 1 2 1 2 1 3 1 4 1 5 CD56-BV421 1 2 1 3 1 4 1 5 CD56-PE/Cy7 1 2 1 3 1 4 1 5 CD56-Pacific Blue Data courtesy of Emily Mace and Jordan Orange, Children s Hospital of Philadelphia. DIC CD56 BV421 Actin Perforin Merge NK NK K562 15 biolegend.com

Brilliant Violet 51 /711 /785 15 Color assay using Brilliant Violet fluorophores Cat. No. Description Clone 317331/2 Brilliant Violet 51 anti-human CD3 OKT3 3143 Brilliant Violet 711 anti-human CD8a RPA-T8 31833 Brilliant Violet 65 anti-human CD14 M5E2 32235 Brilliant Violet 57 anti-human CD19 HIB19 32827/8 Brilliant Violet 65 anti-human CD27 O323 3617/8 Brilliant Violet 421 anti-human CD123 6H6 Brilliant Construction The Brilliant Violet fluorophores do spill over into neighboring channels off the violet laser. However, since spillover values are only one variable that can affect sensitivity in a multicolor panel, intelligently balancing a panel is the most important strategy for the maintenance of resolution in any channel. When two fluorophores exhibit significant spillover and are not of equal brightness, like BV57 and BV65, the best strategy to maintain resolution is to select markers that are not co-expressed on the same cell type, in this instance, CD19 and CD14. Also involved in this balance is the assignment of a very bright fluorophore like BV65 to a lowlyexpressed antigen like CD14, and a dim fluorophore like BV57 to an abundant antigen like CD19. The brightness of BV421 and BV65 and the significant expansion of commercial conjugate choices that are well balanced to antigen expression level make it easier for >12 color panels to become standard in basic and clinical research applications. Cytokine profiling of T cell subsets Cat. No. Description Clone 317331/2 Brilliant Violet 51 anti-human CD3 OKT3 3143 Brilliant Violet 711 anti-human CD8a RPA-T8 32637 Brilliant Violet 785 anti-human CD25 BC96 5331 Brilliant Violet 65 anti-human IL-2 MQ1-17H12 512321/2 Brilliant Violet 421 anti-human IL-17A BL168 52537 Brilliant Violet 65 anti-human IFN-γ 4S.B3 Brilliant Cytokine Staining While quantum dots provide expanded options for colors on the violet laser, they do not perform well for intracellular staining and lack direct conjugate options for intracellular flow cytometry. In contrast to quantum dot nanocrystals, Brilliant Violet -conjugated antibodies are applicable in a 13-color intracellular cytokine and FOXP3 panel for the detection of intracellular antigens (IL-17A, IFN-γ, IL-2), as well as for surface antigens (CD3, CD8, CD25), Fluorophore/PMT BV51 PerCP/Cy5.5 BV711 Alexa Fluor 7 BV785 FITC BV65 PE/Cy7 BV421 APC Specificity CD3 CD4 CD8a CD14 CD25 FOXP3 due to their stability following fixation and permeabilization. Thus, the novel family of Brilliant Violet fluorescent molecules BV421, BV51, BV65, BV65, BV711, and BV785 provide superior new tools for the violet laser line, further enhancing the capabilities for multicolor flow cytometry. PE BV65 APC/Cy7 IL-2 IL-4 IL-17A IL-21 IL-22 IFN-g Live/Dead NIR 16 Customer Service: 858-768-58

Fluorophore/PMT Specificity BV51 CD3 1 5 1 5 Alexa Fluor 7 BV711 PE/Cy5 BV65 APC/Cy7 BV57 PerCP/Cy5.5 CD4 CD8a CD11c CD14 CD16 CD19 CD2 CD3 BV51 1 4 1 3 1 2 1 2 1 3 1 4 1 5 CD19-BV57 CD4 Alexa Fluor 7 1 4 1 3 1 2 1 2 1 3 1 4 1 5 CD8-BV711 BV65 CD27 FITC CD38 PE/Cy7 CD45RA 1 5 1 5 PE BV421 APC PE/CF594 CD56 CD123 CD197 HLA-DR CD14 BV65 1 4 1 3 1 2 CD3 BV51 1 4 1 3 1 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 CD16-APC-Cy7 CD27-BV65 Data courtesy of by Eva Tolosa, University Medical Centre Hamburg-Eppendorf. 17 biolegend.com

Brilliant Violet 57 Intracellular Staining with anti-human IFN-γ Cat. No. Description Clone 52533 Brilliant Violet 57 anti-human IFN-γ 4S.B3 Brilliant Intracellular Staining Intracellular staining of cytokines can be an excellent tool for identifying single cell polarization towards T helper phenotypes. With the recent discovery of many different T helper types, including Th1, Th2, Th17, Tfh, Th9, Th22, and IL-1 producing regulatory T cells, it is critical to delineate T cells using multicolor intracellular flow cytometry. Furthermore, functional characterization of other cell types, such as CD8 + cells, requires the detection of multiple intracellular cytokines. Here, we demonstrate that Brilliant Violet 57 is fully compatible with other anti-cytokine antibodies, including IL-17A Brilliant Violet 421, in a 1-color panel using BioLegend s standard Fixation and Permeabilization buffers. The BV57 antibody is able to clearly resolve the IFN-γ-expressing cells (Th1) from the Th2 and Th17 cells in a mixed human peripheral blood lymphocyte population, after stimulation with PMA/ ionomycin for six hours. Positive gates were determined from FMO and unstimulated controls. The full panel and instrument configuration are shown to the right: (Note: Filter changes and instrument re-configuration may be necessary.) 9 - Color Immunophenotyping infiltrating Lymphocytes in Breast Cancer Tissue Cat. No. Description Clone 3533 Brilliant Violet 57 anti-human CD4 RPA-T4 Brilliant Multicolor A common application in cancer immunology involves profiling primary tumor specimens for tumor infiltrating leukocytes and their proliferation/activation states. Often, these sorts of profiling applications require many concurrent panels to cover phenotypic markers for granulocytes, T cells, B cells, NKT cells, myeloid and dendritic cells. Here, a breast cancer tissue specimen was assayed for T, B and NK cells in a single 9-color panel. Cells were gated on SSC vs. CD45 and then CD3 + populations. Brilliant Violet 57 is an additional marker excited by the violet laser that is brighter than its spectral equivalent, Pacific Orange, and can be used in the same panel as BD Horizon V5 or Live/ Dead Fixable Aqua and QDot 65 in a multicolor panel. In this application, CD4-BV57 is used in place of CD4-QDot 655. The data at right demonstrates the utility of CD4-BV57 as a drop-in replacement, in a 9-color panel. (Note: Filter changes and instrument re-configuration may be necessary.) 18 Customer Service: 858-768-58

Fluorophore/PMT Specificity Laser Voltage Bandpass Dichroic APC IL-2 633 459 67/3 Alexa Fluor 7 CD3 633 385 73/45 69LP BV421 IL-17A 45 368 45/5 Aqua Live/Dead 45 429 525/5 55LP BV57 IFN-g 45 486 585/42 556LP Qdot 65 CD8 45 49 65/4 595LP Alexa Fluor 488 CD45RO 488 489 53/3 55LP PE IL-4 561 419 582/15 PE/Cy5 CD4 561 471 66/2 635LP PE/Cy7 CD45RA 561 524 78/6 75LP 1 5 Th2 1 5 Th17 1 5 Th1 IL-4 PE 1 4 1 3 Th1 IL-17A-BV421 1 4 1 3 Th1 IL-2 APC 1 4 1 3 1 2 1 3 1 4 1 5 IFN-γ BV57 1 2 1 3 1 4 1 5 IFN-γ BV57 1 2 1 3 1 4 1 5 IFN-γ BV57 1 5 1 5 1 5 CD45-APC/eFluor78 1 4 1 3 CD3-PerCP/eFluor71 1 4 1 3 CD2-AF7 1 4 1 3 Fluorophore APC/eFluor 78 PerCP/eFluor 71 efluor 45 BV57 Qdot 65 PE/Cy7 FITC PE/Cy5 Alexa Fluor 7 Aqua Specificity CD45 CD3e HLA-DR CD4 CD8a CD28 NKG2D CD56 CD2 Live/Dead CD4-BV57 1 5 1 4 1 3 1 2 1 3 1 4 1 5 SSC 1 2 1 3 1 4 1 5 CD8-Qdot65 CD4-BV57 1 5 1 4 1 3 1 2 1 3 1 4 1 5 SSC 1 2 1 3 1 4 1 5 CD28-PE/Cy7 CD3-PerCP/eFluor71 1 5 1 4 1 3 1 2 1 3 1 4 1 5 CD56-PE/Cy5 1 2 1 3 1 4 1 5 CD56-PE/Cy5 19 biolegend.com

Brilliant Violet 57 6-Color Treg Identification Cat. No. Description Clone 35137 Brilliant Violet 57 anti-human CD127 A19D5 32629/3 Brilliant Violet 421 anti-human CD25 BC96 Brilliant Clarity Flow cytometry is an effective tool for the identification of Treg (Regulatory T) cells. While FOXP3 is the definitive marker for Tregs, it is an intracellular target that requires fixation and permeabilization of cells for antibody labeling, which precludes researchers from isolating and performing further functional studies on those cells. CD127 and CD25 have been identified as useful cell surface markers for the classification of Tregs without the use of a FOXP3 antibody, characterized as CD127 dim / CD25 + /CD4 + cells. Further characterization of Tregs requires multicolor flow cytometry, potentially with many different labeled antibodies, increasing the need for more fluorophore options. Here, CD127-BV57 was successfully used for PBMC staining with a 6-color panel, including CD25-BV421, to identify Tregs based on cell surface staining. Other functional Treg markers, CD39 and CD73, which are cell surface ectonucleotidase enzymes that contribute to the suppressive capacity of Tregs, were also characterized in this panel. (Note: Filter changes and instrument re-configuration may be necessary.) Brilliant Violet 65 Comparison of Cytokine Expression on CD154 + /CD8 + and CD154 + / CD8 - cells Cat. No. Description Clone 3139 Brilliant Violet 65 anti-human CD8a RPA-T8 31823 Brilliant Violet 421 anti-human CD154 24-31 35965 Brilliant Violet 57 anti-human CD57 HNK-1 32827/8 Brilliant Violet 65 anti-human CD27 O323 Brilliant Stability for Intracellular Staining CD154 (CD4L) is upregulated in activated cells, where it interacts with CD4 to induce B-cell activation, cytokine production and thus, fate determination. For this experiment, T cell activation is determined by expression of IL-2 and IFN-γ. In this analysis, CD3 PerCP/Cy5.5 vs. CD8 BV65 defines CD8 + vs. CD4 + (CD8 - ) T-cells. From those subsets, CD154 BV421 vs. IL-2 Alexa Fluor 7 or CD154 BV421 vs. IFN-γ PE/ Cy7 expression is compared. CD4 + cells express more CD154 and IL-2 than CD8 + cells, and IFN-γ expression is similar in both populations. IL-2 is strongly co-expressed with CD154, especially in CD4 + cells. The majority of IFN-γ in CD8 + cells is singly expressed, while in CD4 + cells, the majority of IFN-γ is co-expressed with CD154, due in part to the reduced CD154 expression in CD8 + cells. However, when comparing the ratio of CD154 + cells to CD154/IFN-γ co-expressing cells, CD154 +, CD4 + and CD8 + T cells are roughly similar in their ability to produce IFN-γ. 2 Customer Service: 858-768-58

Fluorophore BV421 BV57 FITC PerCP/Cy5.5 PE/Cy7 APC Specificity CD25 CD127 CD4 CD3 CD39 CD73 Data provided by Eva Tolosa, University Medical Center Hamburg-Eppendorf. 1 5 1 5 CD4-FITC 1 4 1 3 CD127-BV57 1 4 1 3 1 2 1 3 1 4 1 5 CD3-PerCP/Cy5.5 1 2 1 3 1 4 1 5 CD25-BV421 Data provided by Morgan Reuter and Michael Betts, University of Pennsylvania Data provided by Morgan Reuter and Michael Betts, University of Pennsylvania 1 5 1 5 1 5 CD8-BV65 1 4 1 3 IFN-γ PE/Cy7 1 4 1 3 IL-2 Alexa Fluor 7 1 4 1 3 Fluorophore 1 3 1 4 1 5 CD3 PerCP/Cy5.5 Specificity 1 3 1 4 1 5 CD154 BV421 1 3 1 4 1 5 CD154 BV421 PE/Cy5 PE/Cy5 CD16 CD19 1 5 1 5 FITC PerCP/Cy5.5 AF647 AF7 PE PE/Cy7 BV421 T-bet CD3 Eomes IL-2 Perforin IFN-g CD154 IFN-γ PE/Cy7 1 4 1 3 IL-2 Alexa Fluor 7 1 4 1 3 BV57 BV65 BV65 CD57 CD8 CD27 1 3 1 4 1 5 CD154 BV421 1 3 1 4 1 5 CD154 BV421 Live/Dead Blue 21 biolegend.com

Brilliant Violet 65 Treg Panel using Brilliant Violet fluorophores Cat. No. Description Clone 317435 Brilliant Violet 65 anti-human CD4 OKT4 32629/3 Brilliant Violet 421 anti-human CD25 BC96 3137 Brilliant Violet 57 anti-human CD8a RPA-T8 Brilliant Family Definitive characterization of Treg cells typically requires a combination of cell surface and intracellular staining. Directly labeled antibodies against CD3, CD4, and CD25 are commonly used for cell surface markers on Tregs, while FOXP3 is a specific intracellular marker. Here, we demonstrate the utility of Brilliant Violet fluorophores, BV421, BV57, and BV65, in a Treg staining panel. In the analysis to the right, live cells are positively gated and then analyzed for CD8 vs. CD4 expression. CD4 + cells are then analyzed for FOXP3 vs. CD25. Fluorescence Minus One (FMO) data reveals the specificity of the FOXP3 + CD25 + cells. Being able to utilize such an expanded set of fluorophores greatly expands the options for more complex analysis with 9 or more colors, allowing for more indepth studies on the characteristics and functions of Tregs. Brilliant Violet 65 / Brilliant Violet 65 T cell Activation Panel in Rhesus Macaque Cat. No. Description Clone 3139 Brilliant Violet 65 anti-human CD8a RPA-T8 3241 Brilliant Violet 65 anti-human CD16 3G8 31831 Brilliant Violet 57 anti-human CD14 M5E2 32331 Brilliant Violet 57 anti-human CD2 2H7 35623/4 Brilliant Violet 421 anti-human CD95 DX2 Brilliant Reactivity Rhesus macaque is a common model for immune studies in response to pathogens. In this panel, T-cell activation was assessed in a variety of subsets. A 12 color flow panel was run on an LSRII (45, 488, and 633 nm lasers). On gated lymphocytes, dead cells were excluded, as well as CD16 BV65 and CD14 and CD2 BV57. From the enriched population, T cells were phenotyped into naïve, effector and memory subsets on the total T cell population, as well as broken into CD4 + and CD8 + selective populations by gating on CD28 and CD95. Naïve T cells are CD28 + /CD95 -, memory are CD28 + /CD95 +, and effector T cells are CD28 - /CD95 +. Each T cell subset was then assessed for the degree of expression of activation markers such as CCR5, HLA-DR, CCR7 and proliferation status with Ki-67. 22 Customer Service: 858-768-58

Data provided by Albert Bendelac, Rachel Reboulet and David Leclerc, University of Chicago, and Kelly Lundsten, BioLegend. Fluorophore Specificity PE/Cy7 CD3 BV57 CD8 BV421 CD25 BV65 CD4 Alexa Fluor 647 FOXP3 Count 3 2 Green Live/Dead 1 CD8 BV57 1 5 1 4 1 3 1 1 1 2 1 3 1 4 1 5 1 3 1 4 1 5 CD4 BV65 FOXP3 Alexa Fluor 647 1 5 1 4 1 3 FOXP3 Alexa Fluor 647 1 5 1 4 1 3 Full Panel FOXP3 BV421 FMO CD25 AF647 FMO 1 3 1 4 1 5 CD25 BV421 1 3 1 4 1 5 CD25 BV421 Fluorophore Specificity BV57 CD14/CD2 BV421 CD95 BV65 BV65 PE PE/Cy7 FITC PerCP/Cy5.5 APC Alexa Fluor 7 CD8a CD16 HLA-DR CD28 CCR7 Ki67 CCR5 CD3 CD14/CD2 BV57 1 5 1 4 1 3 CD8 BV65 1 5 1 4 1 3 APC/Cy7 Aqua CD4 Live/Dead 1 3 1 4 1 5 CD16 BV65 1 3 1 4 1 5 CD4 APC/Cy7 1 5 4 4 CD28 PE/Cy7 1 4 1 3 CD28 PE/Cy7 3 2 1 CD28 PE/Cy7 3 2 1 1 3 1 4 1 5 CD95 BV421 1 2 1 3 1 4 1 5 CCR5 APC 1 2 1 3 1 4 1 5 HLA-DR PE 23 biolegend.com

Brilliant Violet Products Available Brilliant Violet 421 Human CD1a HI149 3127 CD1c L161 331525 331526 CD2 TS1/8 39217 39218 CD3 UCHT1 3433 3434 CD4 RPA-T4 3531 3532 CD4 OKT4 317433 317434 CD5 UCHT2 3625 3626 CD8a RPA-T8 3135 3136 CD1 HI1a 312217 312218 CD11b ICRF44 31323 31324 CD11b M1/7 11235 11236 CD11c 3.9 31627 31628 CD14 M5E2 31829 3183 CD16 3G8 3237 3238 CD19 HIB19 32233 32234 CD2 2H7 32329 3233 CD24 ML5 311121 311122 CD25 BC96 32629 3263 CD27 O323 32823 32824 CD28 CD28.2 32929 3293 24 CD31 WM59 33123 CD33 WM53 33415 33416 CD34 561 34369 34361 CD38 HIT2 33525 33526 CD39 A1 328213 CD44 BJ18 33889 33881 CD45 HI3 3431 3432 CD45RA HI1 34129 3413 CD45R/B22 RA3-6B2 13239 1324 CD45RO UCHL1 34223 Brilliant Violet 421 Human CD56 (NCAM) HCD56 318327 318328 CD62L DREG-56 34827 CD64 1.1 3519 352 CD69 FN5 31929 3193 CD73 AD2 3447 3448 CD8 2D1 35221 CD83 HB15e 35323 35324 CD86 IT2.2 35425 35426 CD9 (Thy1) 5E1 328121 328122 CD95 (Fas) DX2 35623 35624 CD15 43A3 323219 CD17a (LAMP-1) H4A3 328625 328626 CD117 (c-kit) 14D2 313215 313216 CD122 (IL-2Rβ) TU27 3399 3391 CD123 6H6 3617 3618 CD127 (IL-7Ra) A19D5 35139 35131 CD134 Ber-ACT35 (ACT35) 3513 CD137 (4-1BB) 4B4-1 39819 CD154 24-31 31823 CD158e1 (KIR3DL1, NKB1) DX9 312713 312714 CD161 HP-3G1 339913 339914 CD183 G25H7 353715 353716 CD184 (CXCR4) 12G5 36517 36518 CD185 (CXCR5) J252D4 356919 35692 CD192 (CCR2) K36C2 35729 CD193 (CCR3) 5E8 31713 31714 CD194 (CCR4) L291H4 359413 359414 CD196 G34E3 35347 35348 CD197 (CCR7) G43H7 35327 35328 CD2 (OX2) OX-14 32929 CD23c (E-NPP3) NP4D6 324611 CD274 (B7-H1, PD-L1) 29E.2A3 329713 For the latest product updates, visit: biolegend.com Customer Service: 858-768-58

Brilliant Violet Products Available Brilliant Violet 421 Human CD279 (PD-1) EH12.2H7 329919 32992 For the latest product updates, visit: biolegend.com CD284 (TLR4) HTA125 312811 CD294 (CRTH2) BM16 35111 35112 CD33 (BDCA-2) 21A 354211 CD34 (Neuropilin-1) 12C2 354513 CD326 (EpCAM) 9C4 324219 CD335 (NKp46) 9E2 331913 CD36 (IL-21R) 2G1-K12 34789 EGFR AY13 352911 GARP (LRRC32) 7B11 35259 HLA-DR L243 37635 37636 IFN-γ 4S.B3 52531 52532 Ig light chain κ MHK-49 316517 316518 IgD IA6-2 348225 348226 IgG Fc HP617 49317 49318 IgM MHM-88 314515 314516 IL-2 MQ1-17H12 5327 5328 IL-4 MP4-25D2 5825 5826 IL-1 JES3-9D7 51421 IL-17A BL168 512321 512322 Ki-67 Ki-67 3555 LAP (TGF-β1) TW4-2F8 349613 Perforin B-D48 35337 T-bet 4B1 644815 644816 TCR α/β IP26 36721 TCR γ/δ B1 331217 331218 Tim-3 F38-2E2 3457 3458 TNF-a MAb11 52931 52932 Brilliant Violet 421 Mouse CD3 17A2 1227 CD3ε 145-2C11 1335 1336 CD4 GK1.5 1437 1438 Brilliant Violet 421 Mouse CD4 RM4-5 1543 1544 CD5 53-7.3 1617 1618 CD8a 53-6.7 1737 1738 CD11b M1/7 11235 11236 CD11c N418 117329 11733 CD16/32 93 11331 CD19 6D5 115537 115538 CD21/CD35 (CR2/CR1) 7E9 123421 CD24 M1/69 11825 CD25 PC61 1233 1234 CD31 39 12423 CD34 MEC14.7 119321 CD41 MWReg3 133911 CD44 IM7 1339 CD45 3-F11 13133 CD45.1 A2 11731 CD45.2 14 19831 CD45R/B22 RA3-6B2 13239 1324 CD48 HM48-1 13427 CD62L MEL-14 14435 CD69 H1.2F3 14527 14528 CD71 RI7217 113813 CD8 16-1A1 14725 14726 CD86 GL-1 1531 CD13 2E7 121421 CD17a (LAMP-1) 1D4B 121617 CD117 (c-kit) 2B8 15827 15828 CD117 (c-kit) ACK2 135123 135124 CD127 A7R34 13523 13524 CD134 (OX-4) OX-86 119411 CD135 A2F1 135313 135314 CD138 (Syndecan-1) 281-2 14257 14258 CD15 (SLAM) TC15-12F12.2 115925 115926 125 µl 5 µl CD152 UC1-4B9 16311 CD183 (CXCR3) CXCR3-173 126521 126522 CD185 (CXCR5) L138D7 145511 145512 CD194 (CCR4) 2G12 131217 25 biolegend.com

Brilliant Violet Products Available Brilliant Violet 421 Mouse (continued) CD196 (CCR6) 29-2L17 129817 129818 CD197 (CCR7) 4B12 12119 1212 CD26 (MMR) C68C2 141717 CD274 (B7-H1, PD-L1) 1F.9G2 124315 CD279 (PD-1) 29F.1A12 135217 135218 For the latest product updates, visit: biolegend.com 26 CD34 (Neuropilin-1) 3E12 14529 CD335 (NKp46) 29A1.4 137611 F4/8 BM8 123131 123132 I-A/I-E M5/114.15.2 17631 IFN-γ XMG1.2 55829 IgM RMM-1 46517 46518 IL-2 JES6-5H4 53825 IL-4 11B11 54119 5412 IL-1 JES5-16E3 5521 IL-17A TC11-18H1.1 56925 KLRG1 2F1/KLRG1 138413 LAP (TGF-β1) TW7-16B4 14147 Ly-6A/E (Sca-1) D7 18127 Ly-6C HK1.4 12831 12832 Ly-6G 1A8 127627 Ly-6G/Ly-6C (Gr-1) RB6-8C5 18433 18434 NK-1.1 PK136 18731 18732 Notch 1 HMN1-12 13615 T-bet 4B1 644815 644816 TCR-β Chain H57-597 19229 TCR γ/δ GL3 118119 TER-119/ErythroidCells TER-119 116233 TNF-α MP6-XT22 56327 Brilliant Violet 421 Rat CD9.1 OX-7 22529 Brilliant Violet 421 Clone Cat. No. Size Secondary Reagents Goat Anti-Mouse IgG Poly453 45317 Goat Anti-Rat IgG Poly454 45414 Donkey Anti-Rabbit IgG Poly464 4641 Streptavidin-BV421 45225 45226 1 μg 1 μl Brilliant Violet 421 Clone Cat. No. Size Isotype Controls Armenian Hamster IgG HTK888 4935 4936 Mouse IgG1, κ MOPC-21 4157 4158 Mouse IgG2a, κ MOPC-173 4259 426 Mouse IgG2b, κ MPC-11 4341 4342 Rat IgG1, κ RTK271 4429 443 Rat IgG2a, κ RTK2758 4535 4536 Rat IgG2b, κ RTK453 4639 464 Brilliant Violet 51 Human CD3 OKT3 317331 317332 CD4 OKT4 317443 317444 CD8a RPA-T8 3147 3148 CD1 HI1a 312219 CD11b ICRF44 31333 31334 CD11c 3.9 31633 CD14 M5E2 31841 31842 CD15 (SSEA-1) W6D3 32327 CD16 3G8 3247 3248 CD19 HIB19 32241 32242 CD2 2H7 32339 3234 CD25 M-A251 356119 CD27 O323 32835 32836 CD28 CD28.2 32935 32936 CD34 581 343527 CD38 HB-7 356611 CD45 HI3 3435 3436 CD45RA HI1 34141 34142 CD49d 9F1 34317 CD56 (NCAM) HCD56 318339 31834 CD69 FN5 31935 CD86 IT2.2 35431 35432 CD9 (Thy1) 5E1 328125 Customer Service: 858-768-58

Brilliant Violet Products Available Brilliant Violet 51 Human CD117 (c-kit) 14D2 313219 CD123 6H6 3621 3622 CD127 (IL-7Rα) A19D5 351331 351332 CD183 (CXCR3) G25H7 353725 CD185 (CXCR5) J252D4 356921 356922 CD194 (CCR4) L291H4 359415 359416 CD196 (CCR6) G34E3 353423 CD197 (CCR7) G43H7 353231 CD279 (PD-1) EH12.2H7 329931 329932 CD335 (NKp46) 9E2 331923 HLA-DR L243 37645 37646 IFN-γ 4S.B3 52543 IgD IA6-2 348219 34822 IgM MHM-88 314521 Ki-67 Ki-67 35517 Brilliant Violet 51 Mouse CD3 17A2 1233 CD4 RM4-5 1553 CD8a 53-6.7 1751 CD11b M1/7 11245 CD11c N418 117337 117338 CD19 6D5 115545 115546 CD25 PC61 1241 1242 CD44 IM7 1343 CD45 3-F11 13137 CD45.2 14 19837 CD45R/B22 RA3-6B2 13247 CD62L MEL-14 14441 CD69 H1.2F3 14531 14532 CD86 GL-1 1539 154 CD9.2 53-2.1 14319 CD117 (c-kit) ACK2 135119 CD15 (SLAM) TC15-12F12.2 115929 CD183 (CXCR3) CXCR3-173 126527 I-A/I-E M5/114.15.2 17635 IFN-γ XMG1.2 55841 Ly-6A/E (Sca-1) D7 18129 Brilliant Violet 51 Mouse Ly-6C HK1.4 12833 Ly-6G/Ly-6C (Gr-1) RB6-8C5 18437 18438 NK-1.1 PK136 18737 18738 TCR-β chain H57-597 19233 19234 For the latest product updates, visit: biolegend.com TER-119/Erythroid Cells TER-119 116237 Brilliant Violet 51 Clone Cat. No. Size Isotype Controls and Secondary Reagents Armenian Hamster IgG HTK888 4941 Mouse IgG1, κ MOPC-21 4171 4172 Mouse IgG2b, κ MPC-11 4345 4346 Mouse IgG2a, κ MOPC-173 4267 Rat IgG1, κ RTK271 4435 Rat IgG2a, κ RTK2758 4547 4548 Rat IgG2b, κ RTK453 4645 Streptavidin-BV51 45233 1 μl Brilliant Violet 57 Human CD3 UCHT1 3435 3436 CD4 OKT4 317445 CD4 RPA-T4 3533 CD8a RPA-T8 3137 CD11b ICRF44 31325 CD14 M5E2 31831 CD16 3G8 3235 CD19 HIB19 32235 CD2 2H7 32331 CD27 O323 32825 CD33 WM53 33417 CD45 HI3 3433 CD45RA HI1 34131 CD45RO UCHL1 34225 CD56 (NCAM) HCD56 318329 CD57 HNK-1 35965 CD127 (IL-7Rα) A19D5 35137 HLA-DR L243 37637 IFN-γ 4S.B3 52533 IgM MHM-88 314517 IL-17A BL168 512323 27 biolegend.com

Brilliant Violet Products Available Brilliant Violet 57 Mouse CD3 17A2 1225 CD4 RM4-5 1541 CD8a 53-6.7 1739 CD11b M1/7 11233 CD11c N418 117331 CD19 6D5 115535 CD44 IM7 1337 CD45 3-F11 13135 CD45.1 A2 11733 CD45.2 14 19833 CD45R/B22 RA3-6B2 13237 CD62L MEL-14 14433 CD9.2 3-H12 15329 Ly-6C HK1.4 12829 Ly-6G 1A8 127629 Ly-6G/Ly-6C (Gr-1) RB6-8C5 18431 NK-1.1 PK136 18733 TCR-β chain H57-597 19231 Brilliant Violet 57 Rat CD9.1 OX-7 22531 Brilliant Violet 57 Clone Cat. No. Size Isotype Controls and Secondary Reagents Armenian Hamster IgG HTK888 4937 Mouse IgG1, κ MOPC-21 4159 Mouse IgG2a, κ MOPC-173 4261 Mouse IgG2b, κ MPC-11 4343 Rat IgG2a, κ RTK2758 4537 Rat IgG2b, κ RTK453 4641 Streptavidin-BV57 45227 1 μl Brilliant Violet 65 Human CD3 OKT3 317321 317322 CD4 OKT4 317437 317438 CD8a RPA-T8 3139 CD1 HI1a 312221 312222 CD11b ICRF44 31331 31332 Brilliant Violet 65 Human CD11c 3.9 31635 CD14 M5E2 31833 CD15 (SSEA-1) W6D3 32331 32332 CD16 3G8 3239 CD19 HIB19 32243 32244 CD2 2H7 32333 CD24 ML5 311123 311124 CD25 BC96 32631 32632 CD27 O323 32829 3283 CD31 WM59 33121 33122 CD34 581 343529 CD38 HIT2 33531 CD45 HI3 3441 3442 CD45RA HI1 34133 CD45RO UCHL1 34237 34238 CD56 (NCAM) HCD56 318333 318334 CD62L DREG-56 34833 34834 CD69 FN5 31937 31938 CD8 2D1 35225 CD86 IT2.2 35429 CD9 (Thy1) 5E1 328127 328128 CD95 (Fas) DX2 35627 CD117 (c-kit) 14D2 313217 313218 CD123 6H6 3625 CD127 (IL-7Rα) A19D5 351333 351334 CD154 24-31 31825 CD161 HP-3G1 339915 CD183 (CXCR3) G25H7 353727 CD184 (CXCR4) 12G5 36521 CD196 (CCR6) G34E3 353419 35342 CD197 (CCR7) G43H7 353223 353224 CD23c (E-NPP3) NP4D6 324619 CD279 (PD-1) EH12.2H7 329923 CD335 (NKp46) 9E2 331925 HLA-DR L243 37639 3764 For the latest product updates, visit: biolegend.com 28 Customer Service: 858-768-58

Brilliant Violet Products Available Brilliant Violet 65 Human IFN-γ 4S.B3 52535 IgM MHM-88 314523 314524 IL-2 MQ1-17H12 5331 IL-4 MP4-25D2 5827 IL-17A BL168 512325 Ki-67 Ki-67 35521 Tim-3 F38-2E2 34517 TNF-α MAb11 52935 Brilliant Violet 65 Mouse CD3 17A2 1237 CD4 RM4-5 1547 CD8a 53-6.7 1743 125 µl CD11b M1/7 11237 125 µl CD11c N418 117333 125 µl CD19 6D5 115539 125 µl CD25 PC61 1235 125 µl CD45 3-F11 13139 1314 CD45.1 A2 11737 125 µl CD45R/B22 RA3-6B2 13243 125 µl CD62L MEL-14 14437 125 µl CD69 H1.2F3 14529 125 µl CD73 TY/11.8 127215 CD8 16-1A1 14729 125 µl CD86 GL-1 1537 125 µl CD9.2 (Thy-1.2) 53-2.1 14317 125 µl CD117 (c-kit) ACK2 135121 135122 125 µl CD127 (IL-7Rα) A7R34 13525 CD138 (Syndecan-1) 281-2 142515 142516 CD15 (SLAM) TC15-12F12.2 115927 125 µl CD183 (CXCR3) CXCR3-173 126523 CD185 (CXCR5) L138D7 145513 CD26 (MMR) C68C2 141721 CD279 (PD-1) 29F.1A12 135219 125 µl F4/8 BM8 123133 IFN-γ XMG1.2 55839 IL-2 JES6-5H4 53829 125 µl IL-4 11B11 54125 IL-17A TC11-18H1.1 56927 125 µl Ly-6A/E (Sca-1) D7 18133 Ly-6C HK1.4 12835 12836 Ly-6G/Ly-6C (Gr-1) RB6-8C5 18439 NK-1.1 PK136 18739 1874 TER-119/Erythroid Cells TER-119 116239 5 μg TNF-α MP6-XT22 56329 For the latest product updates, visit: biolegend.com Brilliant Violet 65 Clone Cat. No. Size Isotype Controls and Secondary Reagents Armenian Hamster IgG HTK888 4943 Mouse IgG1, κ MOPC-21 4161 4162 Mouse IgG2a, κ MOPC-173 4269 Mouse IgG2b, κ MPC-11 4349 435 Rat IgG1, κ RTK271 4433 Rat IgG2a, κ RTK2758 4539 Rat IgG2b, κ RTK453 4649 465 Streptavidin-BV65 45229 1 μl Brilliant Violet 65 Human CD3 OKT3 317323 CD4 OKT4 317435 CD8a RPA-T8 3141 CD11c 3.9 31637 CD14 M5E2 31835 CD16 3G8 3241 CD19 HIB19 32237 32238 CD2 2H7 32335 CD25 BC96 32633 CD27 O323 32827 32828 CD45RA HI1 34135 CD45RO UCHL1 34231 CD56 (NCAM) HCD56 318343 CD62L DREG-56 34831 CD69 FN5 31933 CD8 2D1 35227 CD86 IT2.2 35427 CD123 6H6 3619 CD127 (IL-7Rα) A19D5 351325 351326 CD196 (CCR6) G34E3 353425 CD197 (CCR7) G43H7 353233 CD335 (NKp46) 9E2 331927 331928 IFN-γ 4S.B3 52537 IL-2 MQ1-17H12 5333 TNF-α MAb11 52937 Brilliant Violet 65 Mouse CD3 17A2 1229 125 µl CD4 RM4-5 1545 1546 125 µl CD8a 53-6.7 1741 125 µl CD11b M1/7 11239 125 µl CD11c N418 117339 5 μg 29 biolegend.com

Brilliant Violet Products Available Brilliant Violet 65 Mouse (continued) CD19 6D5 115541 125 µl CD25 PC61 1237 125 µl CD45.1 A2 11735 125 µl CD45.2 14 19835 125 µl CD45R/B22 RA3-6B2 13241 125 µl CD8 16-1A1 14731 CD83 Michel-19 121515 125 µl CD86 GL-1 1535 125 µl CD117 (c-kit) ACK2 135125 5 μg CD138 (Syndecan-1) 281-2 142517 CD15 (SLAM) TC15-12F12.2 115931 CD26 (MMR) C68C2 141723 5 μg IFN-γ XMG1.2 55831 125 µl IL-17A TC11-18H1.1 56929 125 µl Ly-6G/Ly-6C (Gr-1) RB6-8C5 18441 NK-1.1 PK136 18735 TER-119 TER-119 116235 Brilliant Violet 65 Clone Cat. No. Size Isotype Controls and Secondary Reagents Armenian Hamster IgG HTK888 4945 5 μg Mouse IgG1, κ MOPC-21 4163 Mouse IgG2a, κ MOPC-173 4265 Mouse IgG2b, κ MPC-11 4351 Rat IgG1, κ RTK271 4437 Rat IgG2a, κ RTK2758 4541 Rat IgG2b, κ RTK453 4651 5 μg Streptavidin-BV65 45231 1 μl Brilliant Violet 711 Human CD127 (IL-7Rα) A19D5 351327 CD197 (CCR7) G43H7 353227 CD279 (PD-1) EH12.2H7 329927 HLA-DR L243 37643 IFN-γ 4S.B3 52539 IL-17A BL168 512327 Ki-67 Ki-67 35515 T-bet 4B1 644819 Brilliant Violet 711 Mouse CD4 RM4-5 1549 155 CD8a 53-6.7 1747 1748 For the latest product updates, visit: biolegend.com CD11b M1/7 11241 IFN-γ XMG1.2 55835 Ly-6A/E (Sca-1) D7 18131 T-bet 4B1 644819 Brilliant Violet 711 Clone Cat. No. Size Isotype Controls Mouse IgG1, κ MOPC-21 4167 Brilliant Violet 711 Human CD3 OKT3 317327 CD4 OKT4 317439 CD8a RPA-T8 3143 CD11c 3.9 31629 3163 CD56 (NCAM) HCD56 318335 318336 3 CD14 M5E2 31837 CD16 3G8 3243 CD19 HIB19 32245 32246 CD25 BC96 32635 CD27 O323 32833 CD38 HIT2 33527 CD45RA HI1 34137 CD45RO UCHL1 34235 34236 Brilliant Violet, and all Brilliant Violet products are trademarks of Sirigen Group Ltd. Alexa Fluor is a trademark of Molecular Probes, Inc. QDot is a registered trademark of Life Technologies. efluor is a registered trademark of ebiosciences, Inc. Pacific Blue and Pacific Orange are trademarks of Molecular Probes, Inc. BD Horizon is a trademark of Becton, Dickinson and Company. Krome Orange is a trademark of Beckman Coulter, Inc. Customer Service: 858-768-58

Brilliant Violet Products Available Brilliant Violet 785 Human CD3 OKT3 317329 CD4 OKT4 317441 CD8a RPA-T8 3145 CD14 M5E2 31839 CD16 3G8 3245 CD19 HIB19 32239 CD25 BC96 32637 CD27 O323 32831 CD45RA HI1 34139 CD45RO UCHL1 34233 CD127 (IL-7Rα) A19D5 351329 CD196 (CCR6) G34E3 353421 CD197 (CCR7) G43H7 353229 CD279 (PD-1) EH12.2H7 329929 HLA-DR L243 37641 IFN-γ 4S.B3 52541 For the latest product updates, visit: biolegend.com Brilliant Violet 785 Mouse CD3 17A2 1231 CD4 RM4-5 1551 CD8a 53-6.7 1749 CD11c N418 117335 CD19 6D5 115543 CD44 IM7 1341 CD45R/B22 RA3-6B2 13245 IFN-γ XMG1.2 55837 Brilliant Violet 785 Clone Cat. No. Size Isotype Controls Mouse IgG1, κ MOPC-21 4169 Rat IgG2a, κ RTK2758 4545 Rat IgG2b, κ RTK453 4647 BioLegend Brilliant Violet Publications 1. Beres, A.J., et al. 212. J. Immunol. 189:464. 2. Kinsey, G.R., et al. 212. J. Am. Soc. Nephrol. 23: 1528. 3. Xu, Y., et al. 212. J. Immunol. 188:4113. 4. Chattopadhyay, P.K., et al. 212. Cytometry A. 81: 456. 5. Chattopadhyay, P.K., and Roederer, M. 212. Methods 57: 251. 6. Nijnik, A., et al. 212. Blood 119:137. 7. Soghoian, D.Z., et al. 212. Sci. Transl. Med. 4:123ra25. 8. Lemaître, F., et al. 213. J. Immunol. 191: 1578. 9. Covens, K., et al. 213. Blood 121: 5176. 1. Dolfi, D.V., et al. 213. J. Leukoc. Biol. 93: 825. 11. de Silve, T.I., et al. 213. Blood 121: 433. 12. Lawrence, T.M., et al. 213. J. Virol. 87: 5848. 13. Reading, J.L., et al. 213. J. Immunol. 19: 4542. 14. Xu, Y., et al. 213. J. Virol. 87: 376. 15. DeFuria, J., et al. 213. PNAS 11: 5133. 16. Molofsky, A.B., et al. 213. J. Exp. Med. 21: 535. 17. Kim, G., et al. 213. J. Immunol. 19: 151. 18. Leeansyah, E., et al. 213. Blood 121: 1124. 19. Takahashi, H., et al. 212. Stem Cell Trans. Med. 1: 83. 2. Lugli, E., et al. 212. Nature Protocols 8: 33. 21. Alexander, J.J., et al. 212. Kidney Intl. 82: 961. 22. Gatto, D., et al. 213. Nature Immunol. 14: 446. 23. Sage, P.T., et al. 212. Nature Immunol. 14: 152. 24. Baumjohann, D., et al. 213. Nature Immunol. 14: 84. 25. Laurenti, E., et al. 213. Nature Immunol. 14: 756. 26. De Keersmaecker, B., et al. 212. J. Virol. 86: 9351. Scan this QR code to view Brilliant Violet Webinar, Multicolor Flow Cytometry: The Brilliant Violet Family of Fluorophores - Multicolor Qi. Kelly Lundsten Scan this QR code for instant access to our Brilliant Violet Antibodies video tutorial. Scan the QR code for instant access to our Brilliant Violet Antibodies app in itunes. biolegend.com/brilliantviolet_webinar biolegend.com/brilliantviolet biolegend.com/bviossupport 31 biolegend.com

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