Catalog and Reference Manual A Decade of Discovery

Transcription

1 Bı olegend Catalog and Reference Manual A Decade of Discovery

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3 Table of Contents General Information How to Use This Catalog and Reference Manual.... iv Catalog Abbreviations Guide... v Company Profile... vi Ordering Information... viii Custom Solutions Team... x Technical Services... xi Quality and Satisfaction Guarantee... xi Collaborate With Us... xii Terms and Conditions... xii Product Warranties and Use Limitations... xiv Trademarks and Patents.... xv Antibody Product Formats... xvi Frequently Asked Questions.... xvii Technical Charts & Protocols... 1 Products Mouse Immunology Rat Immunology Human Immunology Cytokines, Chemokines, and Related Molecules Mouse Cytokines, Chemokines, and Related Molecules Rat Cytokines, Chemokines, and Related Molecules Human Cytokines, Chemokines, and Related Molecules Bioassay-Validated Recombinant Cytokines Cell Biology Secondary Reagents Isotype Controls Buffers, Solutions and Media Additives Index of Catalog Numbers Master Index BioLegend Worldwide... Inside Back Cover Table of Contents biolegend.com cs@biolegend.com iii

4 How to Use this Catalog and Reference Manual To locate products quickly and easily, this catalog is organized as follows: Main Table of Contents: Products categorized by broad theme or species of interest (e.g., Mouse Immunology) How to Use this Catalog and Reference Manual Sectional Tables of Contents: Products listed alphabetically by product/antigen name (e.g., CD54 or ICAM-1) and by area of biology (e.g., Adhesion Molecules) Note: In the Mouse, Rat, and Human Immunology sections, the first products listed are antibodies to CD antigens, then antibodies are listed alphanumerically by antigen name. Antibodies to TCRs and MHC antigens (Mouse) are listed at the ends of these sections. Master Index: Lists products by common/antigen names, CD name, and clone name. Index of Catalog Numbers: Locates page number via catalog number. For example, to find an antibody specific for mouse TNF receptor, type II, you can locate it several different ways in the Master Index: Antigen common name TNFRII CD name CD120b Clone name TR75-89 Catalog number Once you have found a product of interest, the product pages contain information about both the antigen and the antibody, quality control data, and useful application notes. The small boxes on the bottom of the page describe the format of the antibody/product, the catalog number, the size, and the price, as below. Format Catalog No. Size Price μg $65 Biotin μg $225 Species Reactivity: Listed for clones with multi-species reactivity. Additional information can be found on Cross-Reactivity Charts on pages If you can t find something you are looking for, it is probably already in development! So feel free to contact us: info@biolegend.com. For continually updated product listings, visit BioLegend on-line at biolegend.com. iv Toll Free BIOLEGEND ( )

5 Catalog Abbreviations Guide aa: amino acids AKP: Alkaline phosphatase APC: Allophycocyanin Av: Avidin BA: Bioassay BCA: Bicinchoninic acid protein assay Block: Blocking C : Complement mediated cell depletion CNS: Central nervous system Costim: Costimulatory or costimulation Cy: Cyanine (dye) For your convenience and ease of use, here is a list of abbreviations used throughout this catalog: Deplete or Depletion: Cell depletion ELISA: Enzyme-linked immunosorbent assay ELISA Capture/Detection: Capture or detection reagent for enzyme-linked immunosorbent assay ELISPOT: Enzyme-linked immunosorbent spot assay ELISPOT Capture/Detection: Capture or detection reagent for enzyme-linked immunosorbent spot assay EU: Endotoxin Unit FA: Functional assay FC: Flow cytometry FITC: Fluorescein isothiocyanate GPI: Glycosylphosphatidylinositol HRP: Horseradish peroxidase ICC: Immunocytochemistry ICFC: Intracellular Staining for Flow Cytometry IF: Immunofluorescence microscopy Ig: Immunoglobulin IHC: Immunohistochemistry IP: Immunoprecipitation kd: Kilodalton LEAF : Low endotoxin, azide-free (formulation) LPS: Lipopolysaccharide µg: Microgram mab: Monoclonal antibody MHC: Major histocompatibility complex Neut: Neutralization ng: Nanogram NK: Natural killer cells PBMC: Peripheral blood mononuclear cells PE (or R-PE): Phycoerythrin pg: Picogram SAv: Streptavidin SDS-PAGE: SDS polyacrylamide gel electrophoresis TCR: T cell receptor TNFRSF: TNF receptor superfamily TNFSF: TNF superfamily Treg: T regulatory cells WB: Western blotting Catalog Abbreviations Guide biolegend.com cs@biolegend.com v

6 Company Profile Company Profile BioLegend develops world-class, cutting-edge antibodies and reagents for biomedical research, manufactured in our stateof-the-art facility in San Diego, CA. Our mission is to accelerate research and discovery by providing the highest quality products at an outstanding value, along with superior customer service and technical support. Our product areas include cell immunophenotyping, cytokines and chemokines, adhesion, cancer research, T regulatory cells, stem cells, innate immunity, cell-cycle analysis, apoptosis, and modification-specific antibodies. All of BioLegend s reagents are supported by superior customer service and come with a quality guarantee. Our aggressive product development program, through technology licensing, collaborations, and internal hybridoma development, produces strategic reagents for use in a variety of applications including: Flow cytometry and mass cytometry ELISA and ELISPOT Immunoprecipitation and ChIP Western blotting Immunofluorescence microscopy Immunohistochemistry In vitro and in vivo functional assays BioLegend offers the broadest selection of fluorochrome conjugates for multi-color flow cytometry. With our new Custom Solutions Team, BioLegend is well positioned to offer a wide range of custom products and services tailored to our customers specific needs. Incorporated in June 2002, BioLegend is located in San Diego, in the midst of one of the nation s largest and fastest growing biotechnology clusters. Gene Lay, DVM Founder, President and CEO Co-founder of PharMingen, now BD Biosciences Nan Jiang, Ph.D. Vice President of Operations 20+ years experience in antibody reagent industry, from product development, process development to manufacturing. Craig Monell, Ph.D. Vice President of Sales, Marketing, and Business Development 18 years in the Biotechnology industry, including leadership roles in Marketing, Business Development, R&D, and Project Management at PharMingen/BD Biosciences, Bio- Carta, and Stratagene/Agilent Technologies John Ransom, Ph.D. Vice President of Research and Development 20+ years in Biotechnology, including leadership positions at Advanced Molecular Design, Cytori Therapeutics, Novasite, and Axiom Biotechnologies Kent Johnson Chief Financial Officer Former CFO of PharMingen, now BD Biosciences 19 years reagents manufacturing experience, broad ERP and M&A experience, California licensed CPA Xifeng Yang, M.D. Senior Director of Cell Analysis 20+ years research and development experience in immunology, hematology, and cell biology Shaoquan Ji, DVM/Ph.D. Vice President of Biomarkers and Immunoassays Conducted the development and commercialization of multiplex biomarker immunoassays at LINCO Research/ MILLIPORE Corp. using Luminex xmap technology 25 years experience in the areas of nutrition, physiology, protein chemistry and molecular biology Nancy Huang Senior Director of International Distribution 20+ years experience in Biotech industry in the areas of Marketing/Sales, Distribution Management, Business Development, and Human Resources, including leadership role at PharMingen/BD Biosciences. vi Toll Free BIOLEGEND ( )

7 Worldwide Offices See Inside Back Cover for Contact Information. BioLegend strives to provide quality research reagents and expert technical support to the scientific community worldwide. We look forward to serving your research needs and becoming your long term research partner. Zender-Reinhardt, who are able to discuss projects with customers, help with multi-color flow cytometry panels, and assist in troubleshooting. BioLegend GmbH provides full service sales and applications support in Germany, allowing customers to get their research done efficiently and at a great value. Company Profile BioLegend Japan KK, in partnership with Tomy Digital Biology Co., Ltd., opened in Tokyo in February Yoshiro Namai, Managing Director, and Yoko Yano, Marketing and Business Development Manager, lead a highly experienced and trained technical staff, providing immediate access to BioLegend s entire portfolio of reagents and the highest level of customer service and technical support. BioLegend Europe, established in October 2008, provides convenient ordering and quick delivery for our European customers. We house significant inventory in Europe, allowing for rapid delivery of products to customers throughout Europe and other parts of the world, with minimal lag time. BioLegend UK Ltd.: In May 2010, BioLegend expanded into the United Kingdom and now sells directly to UK customers through a collaboration with Cambridge Bioscience. This collaboration has allowed BioLegend to extend its excellent pricing and value to UK customers. Led by Dr. Vijay Mhaiskar, Managing Director, the BioLegend UK team provides sales and technical applications support for researchers in the UK. BioLegend GmbH: In April 2010, BioLegend officially became a company in Germany, headed by Detlef Reinhardt, Managing Director. He is joined by experienced Scientific Application Manager, Dr. Carsten Wiethe, and Sales Manager, Claudia BioLegend Taiwan: A new R&D facility was opened in Taiwan in biolegend.com cs@biolegend.com vii

8 Ordering Information Ordering Information How to Order from the US and Canada Our Customer Service Department is available 7:00am to 5:00pm (PST), Monday through Friday to take and process your orders. On-line: Visit biolegend.com/ordering Phone/Fax: Phone (Toll-Free US & Canada): BioLegend ( ) Fax: For most efficient order processing, please be sure that the following information is included on your purchase order or correspondence: 1. Name of institution or company 2. Account number (if available) 3. Shipping address 4. Billing address 5. Catalog number, description, and correct price 6. Quantity and size required 7. Name and phone number of purchasing agent 8. Name and phone number of the end user 9. Credit Card number and expiration date or Purchase Order number 10. BioLegend quotation number (if applicable) Shipping from BioLegend All orders are shipped F.O.B. San Diego, California. Shipping and handling charges will be prepaid and added to the invoice. Domestic orders will normally be shipped by overnight air express. BioLegend reserves the right to select the best packaging and shipping method which will ensure the stability of the product and allow efficient order tracking. Our reagents are stable when shipped at ambient temperature. Please store our products under the conditions indicated on the technical information sheet upon arrival. Domestic shipments are sent overnight Monday through Thursday. International shipments are typically sent on Fridays and Mondays. viii Toll Free BIOLEGEND ( )

9 How to Order Internationally Customers located outside of United States may order through BioLegend distributors or through BioLegend directly. We have extensive experience in shipping to many countries directly. We will ship via our preferred courier and the shipping charge will vary depending on the weight of the shipment and the destination country. Customers will be responsible for payment of duties and taxes upon shipment arrival in their respective country. Orders from outside the USA and Canada typically require prepayment before shipping. International Direct Orders: See Inside Back Cover, BioLegend Worldwide, for complete contact Information. Germany: Visit biolegend.com/de and click Kontakt, Ordering for ordering instructions. United Kingdom: Visit biolegend.com/uk and click Contact, Ordering for ordering instructions. Japan: Visit biolegend.com/jp and click お 問 い 合 わせ, Ordering for ordering instructions. Italy: Visit biolegend.com/it and click Contact, Ordering for ordering instructions. All Other Countries: Same as How to Order from the US and Canada, but include your Tax ID number for customs, as well as a copy of the import permit/license (if applicable). Ordering Information International Distributors: Visit biolegend.com/distributors for complete list of Worldwide Distributors. Europe Direct Orders Serviced by BioLegend Europe: For customers located in certain countries in Europe, where BioLegend is selling direct, e.g. Germany, Italy, United Kingdom, or in countries where BioLegend does not have exclusive distributors in place, the orders can be placed with BioLegend Europe via , telephone, or fax. Inquiries: infoeurope@biolegend.com Technical Support: techeurope@biolegend.com Phone/Fax numbers: Visit biolegend.com/contact Shipping from BioLegend Europe BioLegend Europe maintains majority of the catalog items in stock at our Europe warehouse. Orders will be processed and shipped daily if received before 12:00pm. Shipping and handling charges will be prepaid, based on the actual weight of the shipment, and added to the invoice, as applicable. All orders will normally be shipped by overnight air express, unless otherwise specified. BioLegend Europe reserves the right to select the best packaging and shipping method which will ensure the stability of the product and allow efficient order tracking. Our reagents are stable when shipped at ambient temperature. Please store our products under the conditions indicated on the Product Data sheet upon arrival. biolegend.com cs@biolegend.com ix

10 Custom Solutions Team Custom Solutions Team Specialized Products and Services Tailored for Your Research BioLegend s Custom Solutions Team has decades of combined research and manufacturing experience in developing conjugated antibodies, flow cytometry antibody cocktails, ELISAs, multiplex assays, bioassays, recombinant proteins, and custom antibody production. Custom Solutions We will collaborate with you to fully understand your needs, develop a specialized custom solution, and then deliver the highest caliber products and services, along with our quality guarantee. Save time and money by leveraging our expertise. Custom Products Custom antibody cocktails in bulk, including cell surface and intracellular targets Lyophilized antibody cocktails Custom antibody conjugation Multiplex immunoassays on multiple platforms ELISA and ELISPOT kits and sets Antibody screening plates Recombinant protein expression Custom Services Biomarker assay development services Sample analysis for biomarkers Custom manufacturing and packaging Monoclonal and polyclonal antibody production Bioassay development Additional needs? Let s discuss. Custom Value As with all of our products, we provide excellent and reasonable pricing for our custom solutions. Our products and services are backed by our high quality standards, including: Pre- and post-bottling testing Strict documentation and backup of protocols and data Use of regularly calibrated instruments and equipment Lot-to-lot consistency verified by well-defined specifications Highly-skilled personnel with certified biohazard training Daily monitoring of storage refrigerators and freezers Custom Flexibility We understand that each customer has unique project requirements. That s why we are completely flexible with custom manufacturing, packaging, labeling, bulk sizing, batch delivery timelines, etc. We are here to meet your needs. Let s get started on your custom solution us at CST@biolegend.com or call Or contact your local BioLegend representative. x Toll Free BIOLEGEND ( )

11 Technical Services At BioLegend, our Technical Services team is dedicated and trained to answer your questions on our products and their related applications. All of our technical service staff are highly qualified and experienced to address your needs. We welcome any suggestions or comments to help us achieve the highest level of service. For technical support, please call us at (Toll-free US & Canada) or (International), or fax your inquiry to , or send your to: Our Technical Services Department hours are 7:00 am to 5:00 pm, Pacific Standard Time, Monday through Friday. Quality and Satisfaction Guarantee Our strict quality control guidelines allow us to offer a 100% satisfaction guarantee on all our products. As exceptional quality and batch-to-batch consistency are of paramount importance for all of us at BioLegend, each batch must pass stringent biochemical and biological/immunological testing requirements. If, however, any product does not perform as indicated on its Technical Data Sheet, we will either immediately replace it or credit you the original purchase price. Technical Services, and Satisfaction Guarantee Please contact our Technical Services Department if you have any questions or concerns about your product. Our Technical Service Scientists are available to help with assay design, suggest relevant positive controls, and offer troubleshooting tips. Your satisfaction is our number one priority. biolegend.com cs@biolegend.com xi

12 Collaborate with Us Collaborate with Us, and Terms and Conditions BioLegend is committed to providing researchers with the most comprehensive and cutting-edge high quality reagents for life science research. If you have developed reagents or technologies that you feel may be of interest to the life science research community, we would like the opportunity to collaborate with you to commercialize your inventions. We look forward to developing a mutually beneficial collaboration with you. For more information about collaboration and our technology transfer process, please contact Craig Monell, Vice President of Sales, Marketing, and Business Development, at or cmonell@biolegend.com. Pricing Terms and Conditions Prices reflect list price and are subject to change without notice. Payment Methods and Terms We accept credit card purchases on any of our products; please have your VISA, MasterCard, American Express, or Discover Card account number and expiration date available when placing an order. Purchase order numbers are required for all billed shipments. Payment terms are net 30 days. A 1.5% monthly late charge may be charged to accounts that are over 30 days past due. Orders from outside the USA and Canada typically require prepayment before shipping. Limited Use Label License The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer this product its components or materials made using this product or its components xii Toll Free BIOLEGEND ( )

13 Terms and Conditions (continued) to a third party or otherwise use this product or its components or materials made using this product or its components for commercial purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing not to transfer such materials to any third party and to use such transferred materials and/or information solely for research and not for Commercial Purposes. Terms and Conditions Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: use of the product or its components in manufacturing; use of the product or its components to provide a service, information, or data; use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or resale of the product or its components, whether or not such product or its components are resold for use in research If the purchaser is not willing to accept the limitations of this limited use statement, BioLegend requires immediate return of the product with a full refund. Order Confirmation and Duplication Written confirmation of a telephone or fax order is not required. However, if confirmation of an order is sent, it should be clearly marked CONFIRMING ORDER DO NOT DUPLICATE. Duplicated shipments due to incorrectly marked confirming orders will be subject to a Restocking Fee of 20% of the order or minimum of $25, whichever is greater, plus shipping charges. Returns A return authorization number is required on all returns. Please contact BioLegend Customer Service Department to obtain the authorization prior to returning any item. All claims for damaged merchandise during transit must be reported immediately to the shipping carrier, and then to BioLegend. Return authorization requests must be made within 10 working days of order receipt. All claims are subject to investigation before return authorization, replacement or credit can be issued. If BioLegend makes a shipping error, we will replace the product in error or the missing product immediately at no charge. If the customer makes an error on the order and is reported to BioLegend within 10 working days, the customer may obtain return authorization and return the product(s) for partial credit. Upon receipt of authorized returned goods, a credit will be issued, less a 20% restocking fee or minimum of $25, whichever is greater, of the product(s) returned and the original shipping and handling charge. To obtain partial credit, product(s) must be returned within 15 days of receiving return authorization. biolegend.com cs@biolegend.com xiii

14 Product Warranties and Use Limitations Product Warranties and Use Limitations Limited Warranty and Liability Please review specifications of the product as published in the product details section of our website or call and request a copy from our Technical Service Department before ordering. Product details on the website generally reflect the applications and conditions for use. Products should be used in accordance with the technical information sheet shipped with the product. BioLegend reserves the right to change product specifications without prior notice. All products supplied by BioLegend are warranted, to the original purchaser only, to conform to our specifications provided with the shipment. This warranty does not extend to any product that has been modified in any way by person(s) other than BioLegend personnel or to any products that are handled contrary to the technical information sheet. Should any product fail to perform as warranted, upon notification to our Technical Service Department and investigation, BioLegend shall promptly replace it at no charge. Our obligation and your only remedy is limited to such replacement of the product or a full credit of the original purchase price of the product. BioLegend reserves the right to discontinue sales of any products or to change product descriptions and formulations at any time without prior notice to purchasers. BioLegend does not make any other warranty or representation whatsoever, whether express or implied, with respect to these products. BioLegend does not make any warranty of suitability, non-patentinfringement, merchantability or fitness for any particular research use or other purposes of any product. The entire risk as to the performance of this product is assumed by buyer. In no event shall BioLegend be liable for incidental, consequential or punitive damages. Product Use Limitations Unless otherwise clearly indicated on product labels, BioLegend products are FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE xiv Toll Free BIOLEGEND ( )

15 Trademarks and Patents Trademarks BioLegend, BioLegend Logo, LEAF, The path to legendary discovery, TruStain FcX, ELISA MAX, and LEGEND MAX are the trademarks and property of BioLegend, Inc. ABTS is a registered trademark of Boehringer Mannheim GmbH. BD, BD FACSCalibur, BD FACSCanto, BD FACScan, BD FACSVantage SE, BD FACSAria, BD LSRFortessa, BD CyChrome, BD Influx and BD Horizon are trademarks of Becton, Dickinson and Company. Brilliant Violet, Brilliant Violet 421, BV421, Brilliant Violet 570, and BV570 are trademarks of Sirigen Group Ltd. Cascade Blue, Alexa Fluor, Texas Red are registered trademarks and Pacific Blue, Pacific Orange, and Cascade Yellow are trademarks of Molecular Probes, Inc. Coulter and EPICS are registered trademarks and XL, XL- MCL, CyAn, Gallios, and MoFlo are trademarks of Beckman Coulter, Inc. Patents Alexa Fluor and Pacific Blue : U.S. Patent Nos. 5,696,157; 6,130,101; 5,798,276; 5,830,912; 6,562,632B1; 6,716,979 pending patent applications and corresponding foreign equivalents, owned by Invitrogen Corp. Brilliant Violet : U.S. Patent Nos. 7,914,984; 7,811,755; 7,666,594; 7,629,448; 7,214,489; 7,270,956; 7,144,950; 7,897,684. Licensed from Sirigen, owned by The Regents of the University of California. Allophycocyanin (APC) conjugates: U.S. Patent No. 5,714,386 Cyanine (Cy): U.S. Patent Nos. 4,981,977; 5,268,486 and other patents pending. R-Phycoerythrin (R-PE) and Allophycocyanin (APC): U.S. Patent Nos. 4,542,104 and 5,714,386 PE-Cy7: U.S. Patent No. 4,542,104 APC-Cy7: U.S. Patent No. 5,714,386 Peridinin chlorophyll protein (PerCP): U.S. Patent No. 4,876,190 CD44: U.S. Patent No. 5,108,904 CD62P: U.S. Patent No. 4,783,330 Antibody to Human Fusin (clone 12G5): U.S. Patent No. 5,994,515 Costar is a registered trademark and Stripwell is a trademark of Corning, Inc. Cy, including Cy3, Cy5, Cy5.5 and Cy7, is a trademark of Amersham Biosciences Ltd. CyAn ADP and MoFlo are trademarks of Dako Denmark A/S. CyTOF is a registered trademark of DVS Sciences. DyLight is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries. FLAG is a registered trademark of Sigma-Aldrich Co. influx is a trademark of Cytopeia, Inc. Synergy and Eclipse are trademarks and Reflection is a registered trademark of icyt Mission Technology, Inc. C6 Flow Cytometer is a registered trademark of Accuri Cytometers, Inc. ImageStream is a registered trademark of Amnis Corporation. MACSQuant is a trademark of Miltenyi Biotec Inc. 12G5: U.S. Patent No. 5,994,515 Antibodies to Human IL-4: U.S. Patent No. 5,041,381 Antibodies to Human and Murine IL-10: U.S. Patent No. 6,239,260 Antibodies to vil-10: U.S. Patent No. 5,756,342 Antibodies to Human and Murine IL-10 Receptor: U.S. Patent No. 5,863,796 Antibodies to Human and Murine IL-17: U.S. Patent No. 6,274,711 Antibodies to Human GM-CSF: U.S. Patent No. 5,070,013 Antibodies to Human and Murine SLAM: U.S. Patent No. 5,576,423 Antibodies to Mouse IL-6 Receptor, Mouse IL-9, Human IL-9: U.S. Patent No. 5,157,112; European Patent No Antibodies to Human IL-9 Receptor: U.S. Patent No. 5,962,269; European Patent No Antibodies to Human and Murine CD317: U.S. Patent No. 7,049,408 Annexin V: U.S. Patent No. 5,834,196 Antibodies to T-bet: U.S. Patent Nos. 7,745,581; 7,393,944; 7,365,169 Antibodies to p27kip1: U.S. Patent Nos. 5,688,665; 6,635,450; 6,242,575; 6,355,774 Antibodies to Human Phospho-ATM (Ser1981): U.S. Patent No. 7,108,992 Antibodies to Gamma-H2A: U.S. Patent Nos. 6,362,317; 6,884,873 Antibodies to Human KIR2DL4: U.S. Patent No. 7,435,801 Trademarks and Patents biolegend.com cs@biolegend.com xv

16 BioLegend Antibody Formats BioLegend Antibody Formats Format Purified LEAF Purified* Biotin Brilliant Violet 421 Brilliant Violet 570 Application FC, ELISA Cap., WB, IP, IHC, IF Bioassays, ELISPOT Cap., Blocking/Neutralization FC, ICFC, ELISA Det., ELISPOT Det., IHC, IF FC, ICFC, IF FC, ICFC Format Tandem Dyes PE/Cy5 (Cy-Chrome ) PE/Cy7 PerCP/Cy5.5 APC/Cy5.5 APC/Cy7 Application FC FC FC, ICFC FC FC FITC PE APC Alexa Fluor 488 Alexa Fluor 647 Alexa Fluor 700 FC, ICFC, IF FC, ICFC FC, ICFC FC, IF FC, IF FC Applications key: FC: Flow Cytometry ELISA Cap./Det.: ELISA Capture/ Detection ELISPOT Cap./Det.: ELISPOT Capture/Detection WB: Western Blotting IP: Immunoprecipitation ICFC: Intracellular staining for Flow Cytometry IHC: Immunohistochemistry IF: Immunofluorescence Microscopy Pacific Blue FC, IF PerCP FC, ICFC Cy3 IF DyLight 405 FC, IF DyLight 488 FC, IF DyLight 594 FC, IF DyLight 649 FC, IF *BioLegend s LEAF (Low Endotoxin, Azide-Free) purified antibodies are specifically designed for functional analyses, providing the most accurate results with minimal negative effects. LEAF purified antibodies contain no preservative, are 0.2 µm filter sterilized, tested for sterility, and endotoxin level is <0.1 EU/µg of the protein (<0.01 ng/µg of the protein) as determined by the LAL test. xvi Toll Free BIOLEGEND ( )

17 Frequently Asked Questions General 1. What is the shelf life of BioLegend products? Most of our products (except some buffers or unless stated otherwise) have a guaranteed shelf life of one year from date of receipt by the end-user, under proper storage and handling conditions as instructed on our Product Data Sheets. For recombinant proteins, the minimum guaranteed shelf-life is 6 months from the date of receipt by the end-user. 2. What are the concentrations of your antibodies? For antibodies offered in µg sizes, concentrations are typically (exceptions for some products) as follows: APC, APC/Cy5.5, APC/Cy7, PE, PE/Cy5, PE/Cy7, PerCP, PerCP/Cy5.5 Purified, Biotin, FITC, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, Pacific Blue LEAF Purified 0.2 mg/ml 0.5 mg/ml 1.0 mg/ml For the antibodies offered per test, per plate, or per Western blot, concentrations vary, depending on optimal titer in the assay. If you have the Product Lot#, use our Concentration Lookup tool at biolegend.com/concentrationlookup or contact our Technical Services at or techserv@biolegend.com. 3. In what formats and for what applications do you offer your antibodies? See page xvi for list of all BioLegend antibody formats and applications. 4. Multiple clones are available for a particular specificity. How do I choose the right one? Check the Product Data Sheets on-line to review applications/characteristics of each clone. If you are not looking for a specific clone, we recommend using the most popular clone based on PubMed or Google Scholar. This will give you an idea on how often a clone is used compared to another. If you still have questions, please contact BioLegend Technical Services at: Toll Free Phone (U.S., Canada): Phone: Fax: techserv@biolegend.com 5. How do I obtain a Material Safety Data Sheet (MSDS) for your products? On each product data sheet there is a link to all of the available MSDS. Or check our website under Support. If your product isn t covered in this section, please contact BioLegend Technical Services. 6. What is BioLegend s Quality Policy? 100% Satisfaction Guarantee. If BioLegend s product does not perform as described on its product data sheet, we ll replace it or refund 100% of the original purchase price. 7. What is BioLegend s Return Policy? All claims for merchandise damaged during transit must be reported immediately to the shipping carrier, then to BioLegend. For all other returns, return authorization is required, and requests must be made within 10 working days of order receipt. Obtain a return authorization number from our Customer Service department. All claims are subject to investigation before return authorization, replacement, or credit can be issued. If your order was placed incorrectly, the product(s) may be returned for partial credit, and the return must be postmarked within 15 days of the return authorization date. Upon receipt of authorized returned goods, a credit will be issued, less a 20% restocking fee or $25, whichever is greater, and the shipping and handling charge. 8. Does BioLegend manufacture antibodies according to GMP guidelines? Although BioLegend does not manufacture antibodies in a certified GMP facility, our products are manufactured according to GMP guidelines. For more information, visit our Quality Control page at biolegend.com/qc. 9. What is the epitope of the antibody? BioLegend does not epitope map antibodies. Sometimes this information is indicated on the data sheets, if published, but generally, we will not know the exact epitope of the antibody. Frequently Asked Questions biolegend.com cs@biolegend.com xvii

18 10. How do I look for reagents that are crossreactive with a different species (e.g. Rhesus or Cynomolgus)? See Cross-Reactivity Charts on pages You may also go to biolegend.com and perform an Advanced Search, which contains a drop-down menu for Species Reactivity, or visit our Non-Human Primate page at biolegend.com/nhp. Frequently Asked Questions Ordering 11. What is the advantage of ordering per test, plate, or µl instead of per mg? Antibodies offered per test, plate, or µl have been pretitrated for optimal performance in flow cytometry, ELISA, or Western Blot, respectively. Pre-titrated antibodies provide greater convenience, with savings of time and reagents because antibody titration is not required. Note, a particular antibody packaged in a μg format (untitrated) is not necessarily from the same batch number as the test format (pre-titrated) of that antibody, but both formats must pass the same QC testing criteria. Please also note that the test format of an antibody does not always have a lower concentration than the μg format; this information is batch-specific. 12. How are BioLegend reagents shipped? Most products are shipped at ambient temperature. Our products are sufficiently stable to maintain optimal performance after overnight shipping. 13. When can I expect to receive my order if I order directly from BioLegend? For U.S. and Canada orders placed by 3 pm PST Mon- Thur, if products are in-stock, they are shipped the same day, via Federal Express overnight, so you can expect to receive the products the next day. International direct orders are shipped on Mondays and Fridays, and typically arrive within 2 10 days, depending on how long they are in customs. 14. Why should I order from an international distributor? For direct orders shipped outside the U.S., shipping costs typically start at USD $50 for antibody orders and $100 for dry ice shipments. This may vary country to country. Additionally, prepayment is typically required for direct international shipments, and taxes and duties will be paid by the recipient upon shipment delivery. In some countries, the customer needs to apply for an import permit. When ordering from a distributor, the payment terms are more flexible, the customer and technical support is more immediate, and the customer has greater convenience in not having to deal with customs agencies, duties/taxes, and currency exchange. Flow Cytometry 15. What is the difference between test and µg sizes? The test size products are pre-titrated for optimal staining of 1 million cells in 100 µl volume. The µg size products are at a defined concentration regardless of the optimal usage. Regardless of format, it is very important to use the correct isotype control at the same amount (µg) as the antibody of interest. If the concentration of the antibody is not on the vial, then use our Concentration Lookup tool at biolegend.com/ concentrationlookup or contact our Technical Services at or techserv@biolegend.com. 16. What is the difference between the FC and ICFC formulations? Do they have different optimal concentrations of utilization? FC is for cell surface staining, and ICFC is for intracellular (IC) staining. Yes, the concentrations can be different. The ICFC antibodies and isotypes have optimized fluorophore conjugation properties to give optimal performance for IC staining. 17. Can I use less volume or fewer cells than the recommended amount? Yes, you can use fewer cells or less volume per test, as long as the antibody concentration does not change. For example, if you decrease the staining volume from 100 to 50 µl, you can use half the amount of antibody. But, if you decrease your cell numbers from 1 million to 100,000 cells and your staining volume is still 100 µl, then you should still use the data sheet recommended amount of antibody. 18. How does BioLegend choose the optimal concentration of an antibody in flow cytometry? Generally, we test 4 to 6 dilutions with the most commonly used target cells for the titration curve, then determine the optimal concentration based on the S/N (Signal/Noise) ratio. We choose the concentration before the S/N decreases, so the optimal concentration is at its saturation, if possible. 19. Can I use the antibody at a lower concentration than the recommended amount? Possibly; we always recommend performing a titration. Sometimes less antibody is suitable for your experimental needs, particularly if antibody saturation in your staining is not a concern. The % positive staining should be maintained at your selected concentration; i.e., if while you are titrating, the population % drops, then you are not staining all the cells and should use a higher concentration. xviii Toll Free BIOLEGEND ( )

19 20. Can I use BioLegend antibodies for cell sorting? Yes, in general, our antibodies can be used for cell sorting, but we do not routinely test this in-house. Please note, the fluorophore conjugates are not endotoxin tested and contain 0.09% azide. Typically, this low amount of azide will not affect viability of the cells. As for activation of cells, typically, the incubation time with the antibodies for staining is short (~20 minutes), and this should not cause activation. Incubation of antibodies with cells at 4 C or on ice will help prevent activation. 21. What is the F/P ratio for my fluorophoreconjugated antibody? All antibodies labeled with PE, APC and their tandem dyes have a F/P of 1:1. Other formats, such as FITC or Alexa dyes, have various F/P ratios, typically within the range of Will the 0.09% sodium azide in my fluorophoreconjugated antibody affect the viability of my flow sorted cells? Diluting the antibodies for staining also dilutes the sodium azide to a concentration likely not to be toxic to the cells. Since the cells are left in contact with the antibody (and the azide) for only 1/2 hour, then washed, there will be little or no residual azide in the sorting buffer, so the chances of residual toxicity are very low (per the Purdue Cytometry website). 23. Which FOXP3 clone should I use? For detecting human FOXP3, based on our internal testing data, both 259D and 206D clones give good staining. Please see the Treg homepage for the differences between clone 206D or 259D. 206D recognizes human FOXP3 epitope in the region of amino acids The 259D antibody recognizes human FOXP3 epitope in the region of amino acids In the following publication the authors state 206D clone is superior to FOXP3 antibody clones 236A/E7 and PCH101. However, they did not compare with clone 259D. Grant, J., et al. Cytometry B Clin Cytom Mar; 76(2): Both of the antibodies perform well in our hands and we do not have a particular preference. However, in the current literature, scientists seem to prefer 206D. For other mammalian species, 150D is recommended. 24. Can FOXP3 be co-stained with IFN-γ and surface markers? We have tested IFN-γ with FOXP3 staining and it does work. It is recommended to use BioLegend s FOXP3 Fix/Perm Buffer set and the FOXP3 staining protocol. The only addition is to add the cytokine antibody at the same time as the FOXP3 antibody. In order to capture cytokines intracellularly, cells should be stimulated in the presence of monensin or brefeldin A. The Intracellular Staining Guide on page ### is a useful reference for generating various cytokine producing target cells. 25. What is the most common reason for lack of IL-17 staining? Most failures of IL-17 I/C staining are due to a stimulation problem. PMA/Ionomycin in liquid form is very unstable. We recommend storing single use aliquots at -20 degrees for no longer than 6 months. The aliquots should be thawed at ambient temperature, and diluted in PBS to a working concentration immediately before stimulation. Brilliant Violet 26. What is Brilliant Violet? Brilliant Violet is family of fluorescent polymers created by Sirigen based on Nobel Prize winning chemistry. The first in the series is Brilliant Violet 421, excited by the violet laser at 405 nm and emitting optimally at 421 nm, which can be used in place of Pacific Blue, Horizon V450, efluor 450, and Alexa Fluor 405. Brilliant Violet 570 is also excited by the violet laser, emitting optimally at 570 nm, and can be used in place of Pacific Orange, depending on the filter used. 27. How does Brilliant Violet perform compared to other common fluorophores? Brilliant Violet 421 antibody conjugates give an exceptional signal-to-noise ratio, in some cases greater than 10-fold better than Pacific Blue. It gives PEequivalent brightness or better on the violet laser. Brilliant Violet 570 antibody conjugates also provide much improvement over Pacific Orange, by as much as 6-fold. 28. Can Brilliant Violet be used for microscopy? Yes, Brilliant Violet 421 is exceptionally bright and photostable, making it an excellent choice for microscopy. Brilliant Violet 570 has not been fully validated for microscopy. 29. How can I be sure Brilliant Violet will work for me? Select Brilliant Violet antibodies have been betatested and verified by over 20 premiere immunology research labs world-wide, in a variety of applications including multi-color flow cytometry, microscopy, tetramer staining, and cell sorting, as well as a variety of instruments, including common flow cytometers and the Amnis ImageStream. BioLegend QC tests every lot of product pre- and post-bottling. As with all products, we back every Brilliant Violet product with our 100% guarantee. Frequently Asked Questions biolegend.com cs@biolegend.com xix

20 Frequently Asked Questions 30. What bandpass filter should I use to detect Brilliant Violet 421 and Brilliant Violet 570? We recommend using the standard 450/50 filter to detect Brilliant Violet 421, which is the same filter used to detect Pacific Blue. For Brilliant Violet 570, we recommend the 585/42 bandpass filter, commonly used for Pacific Orange. Other filters can be utilized, depending on the panel of fluorophores used. See our on-line Fluorescence Spectra Analyzer for complete excitation and emission data. 31. Will Pacific Blue antibodies be discontinued? Most Pacific Blue antibodies will not be discontinued, and we will continue to make them available to customers. As more customers transition to Brilliant Violet, discontinuation will be based upon customer demand for products. Functional Antibodies 32. I am going to do a functional assay. Which grade of purified antibody should I choose? BioLegend s LEAF (Low Endotoxin, Azide-Free) purified antibodies are specifically designed for functional analyses, providing the most accurate results with minimal negative effects. LEAF purified antibodies are tested for sterility, and endotoxin is <0.01 ng/µg of antibody. 33. Can I get a discount for a bulk antibody purchase? Yes, we do provide discounting based on quantity of antibody requested. Please contact us at sales@biolegend.com to obtain a quote. 34. Does BioLegend test each LEAF antibody by functional assay? No, BioLegend does not test LEAF antibodies by functional assay. Due to the complexities and varying uses of biofunctional antibodies, and because of the large product portfolio, BioLegend does not currently perform functional assays as routine antibody QC. However, we do provide references in which the antibodies were used for functional assays, and we perform QC to verify the specificity and quality based on our strict specifications. 35. Does BioLegend test each LEAF antibody for potential pathogens? No, BioLegend does not test for pathogens in-house. However, we can recommend an outside vendor to perform this testing as needed. Isotype Controls 36. How do I choose the right isotype control for the primary antibody I m using? Refer to the listed isotype and format of each primary antibody, and match the isotype control appropriately. For example, if you have a PE conjugated primary antibody with Rat IgG1, k isotype, use PE Rat IgG1, k isotype control Catalog # or as your isotype control. For your convenience, most products have their isotype control listed under their related products on our website. 37. How do I know how much isotype control to use for test size products? Match your isotype to the concentration of the primary antibody. If your vial label does not contain the antibody concentration, call or tech support and ask for the concentration of your lot of antibody. If you have the Product Lot#, use our Concentration Lookup tool at biolegend.com/concentrationlookup or contact our Technical Services at or techserv@ biolegend.com. ELISA 38. What is the difference between LEGEND MAX, ELISA MAX Deluxe, and ELISA MAX Standard? LEGEND MAX Kits contain Pre-coated Plates and all necessary reagents, ready to use. Most convenient, shortest protocol, full analytical and biological validation. We recommend LEGEND MAX Kits for users who are new to ELISAs or are short on time. ELISA MAX Deluxe Sets contain Uncoated Plates, Pre-titrated Capture and Detection Antibodies, Avidin- HRP, Recombinant Standard, Coating Buffer, Assay Diluent, and TMB Substrate Reagent. Convenient and costeffective option. ELISA MAX Standard Sets contain Pre-titrated Capture and Detection Antibodies, Recombinant Standard, and Avidin-HRP. Most cost-effective, plates not included, optimization required. 39. Can I mix reagents from different ELISA MAX Sets, or use them for other applications? This is not recommended. The ELISA MAX reagents are optimized for a particular set lot, and they are not recommended for applications other than ELISA. 40. How should the plates be stored after coating? If coated plates cannot be used immediately, they should be sealed and stored overnight at 4 C. 41. What kind of coating buffer should I use for my ELISA? This may be dependent on the cytokine being detected, but in general, scientists use PBS, ph 7.4 or carbonate buffer at ph 9.5. xx Toll Free BIOLEGEND ( )

21 42. Why do you recommend not to use sodium azide in ELISA buffers? Azide is an irreversible inhibitor of horseradish peroxidase (HRP). 43. Is there a time frame in which the coated ELISA plates should be used? The plates can be coated with the capture antibody and stored sealed at 4 C for 2-3 days. During the blocking step, the plate can be stored in blocking buffer for 1 2 days at 4 C (sealed), then continue with rest of the protocol. The plates should not be allowed to dry. 44. Where can I find a troubleshooting guide for ELISA? biolegend.com/elisa_troubleshooting 45. Can I use components from different companies for my ELISA? No, this is not recommended. Different kits utilize different antibodies, and antibody specificity partially determines how much signal is detected. The recombinant standards in different kits also vary; some are expressed and purified by different methods. Even expressed from the same source (e.g., E. coli), recombinant proteins can show greater than 10-fold differences in immunoreactivity from lot to lot, primarily due to refolding inconsistency. Different kit standards may also be produced and calibrated against different references. There is currently no universally accepted standardization for cytokine immunoreactivities. Each BioLegend ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness. Any changes to the reagents (standards, antibodies, matching matrices) or protocol will affect the final assay performance. 46. How can I obtain better ELISA signal and sensitivity? Increase incubation times (1st incubation, detection, avidin-hrp or TMB substrate) Shake plates during incubation steps Make sure that the standard is completely reconstituted before use. Increased washing and soaking in between washings to further decrease background. If possible, read the plate at nm for background subtraction. Improve reproducibility by controlling pipetting error or washing with a larger volume of wash buffer. If available with your software, use 5-PL or 4-PL curve-fitting (instead of linear curve-fitting) for better calculation at the lower end of the curve. 47. Should serum or plasma samples be used in my ELISA experiment? This depends on the targets being detected and the biological processes. Customers are advised to study the difference between serum and plasma for the targets of interest and decide on the sample type to be used for quantification. Depending on target, there may be a difference in concentration between serum and plasma. In general, plasma/serum samples should be free of particulate matter, contain no excess lipids, and have no hemolysis. These types of contaminants will contribute to background and adversely affect the precision of the assay. To ensure precise measurements, the most important factor is consistency in preparation of plasma or serum samples. That is, centrifuge samples at the same speed, for the same amount of time, remove the serum or plasma immediately after centrifugation, and aliquot and freeze the samples for the same amount of time. Avoid repeated freeze-thaw cycles. 48. Why are my samples cytokine concentrations different when tested in LEGEND MAX Kits vs. other ELISA Kits? Cytokine kits from different manufacturers do quantitate samples differently at the pg/ml level. The immunoreactivity of reagents vary among different manufacturers, and for some cytokines, concentrations can vary a few-fold. When quantifying cytokines at the picogram level, the general consensus is that it is more important to have matching biological trends than matching absolute concentrations. So the same cytokine measured in different vendors kits should show the same pattern of biological changes predicted for the relevant biological treatment. It is critical for kits produced by the same vendor to keep its assay products consistent from lot to lot over time. Our scientists tested ELISA standards from competitors kits, and results showed clear discrepancies in the relative potency of the standards in terms of their immunoreactivity. Western Blot 49. What are possible causes of High Background in my Western Blot? Transfer buffers may have become contaminated. Post-antibody washes may not have been performed with sufficient time or volume. Blocking and incubation agents were not freshly prepared or were too dilute. Frequently Asked Questions biolegend.com cs@biolegend.com xxi