140-004-047.02 1. The isolation of CD21 of isolation The 1.1 1. Description 4.

Contents 2. Capacity Components 3. CD21 which is placed in the magnetic field of a MACS Separator. The Separator. MACS of a field magnetic the in placed is which conjugated antibodies, as primary labeling reagent, reagent, labeling primary as antibodies, conjugated Product format Biotin MicroBeads, as secondary labeling reagent. In between the the between In reagent. labeling secondary as MicroBeads, Biotin of biotin- acocktail with labeled magnetically indirectly are B cells macsde www.miltenyibiotec.com www.miltenyibiotec.com are subsequently depleted by separation over a MACS® Column, Column, over a MACS® by separation depleted subsequently are Storage two labeling steps no washing steps are required are steps no washing steps labeling two transitional the and B-1 cells, the non-B cells, the First, procedure. Phone Phone Friedrich-Ebert-Straße Friedrich-Ebert-Straße Miltenyi Biotec B.V. KG &Co. Biotec Miltenyi

Protocol Description References Example of a separation using the Marginal Zone and and Zone Marginal the using of aseparation Example Principle of the MACS® Separation MACS® of the Principle cells cells 2.5 2.4 2.3 2.2 2.1 1.4 Applications 1.3 1.2 1.1 Follicular Isolation Kit Isolation BCell Follicular int +49 2204 8306-0, 2204 +49 @ CD23

miltenyi.com

Magnetic labeling of Bcells FO labeling Magnetic separation Magnetic labeling Magnetic preparation Sample requirements instrument and Reagent information Background Separation MACS® of the Principle Magnetic separation: Positive selection of CD23 selection Positive separation: Magnetic hi

follicular (FO) B cells is performed in a two-step atwo-step in performed is (FO) Bcells follicular

For 2×10⁹ total cells, 2×10⁹For total at 2–8 light from Store protected 2×2 mL Anti-BiotinMicroBeads: mL 2×2 2 mL CD23 MicroBeads, mouse: MicroBeads, CD23 1 mL All components are supplied in buffer buffer in supplied are components All Cocktail, mouse: mouse: Cocktail, vial label. vial CD43, CD4, CD93, and Ter119. and CD93, CD4, CD43, MicroBeads conjugated to monoclonal anti- monoclonal to conjugated MicroBeads anti- monoclonal to conjugated MicroBeads containing stabilizer and 0.05% sodium azide. 0.05% sodium and stabilizer containing freeze. The expiration date is indicated on the on indicated is date expiration The freeze. IgG2a). rat (isotype: antibody CD23 mouse against antibodies anti-mouse monoclonal biotin antibody (isotype: mouse IgG1). mouse (isotype: antibody biotin 68, 5142968, Fax Fax mL MZ & FO B Cell Biotin-Antibody Biotin-Antibody BCell &FO MZ mL +49 2204 85197 2204 +49 hi CD23 Bergisch Gladbach, Germany Bergisch Gladbach, of non-MZ and FO Bcells FO and of non-MZ : Depletion of non-MZ and FO B FO and of non-MZ : Depletion low/– marginal zone (MZ) and and (MZ) zone marginal Cocktail of biotin-conjugated of biotin-conjugated Cocktail up to 20 separations. 20 to up .

The labeled cells cells labeled The

°C. Do not Do °C. and Anti- and

+

FO B FO mouse Kit CellB Isolation Follicular Zone and Marginal The Marginal Zone and Follicular B Cell Isolation Kit has been been has Kit Isolation Cell B and Follicular Zone Marginal The Applications 1.3 1.2 page 1/5 ● C57BL/6 mice. mice. C57BL/6 directly labeled with CD23 MicroBeads in a second step and and step asecond in MicroBeads CD23 with labeled directly are Bcells FO the subsets two these separate further to order In developed for the isolation of marginal zone (MZ) B cells and and Bcells (MZ) zone of marginal isolation for the developed unlabeled fraction contains the pre-enriched MZ and FO Bcells. FO and MZ pre-enriched the contains fraction unlabeled magnetic field, the magnetically retained FO B cells can be eluted eluted be can cells FO B retained magnetically the field, magnetic follicular (FO) B cells from of untreated BALB/c and BALB/c and of untreated spleens from (FO) Bcells follicular as the positively selected cell fraction. cell selected positively the as the enriched MZ B cells. After removing the column from the from column the removing After Bcells. MZ enriched the contains through The flow Separator. MACS of a field magnetic the isolated by separation over a MACS Column, which is placed in in placed is which Column, over aMACS by separation isolated

1. 1. 1.

2. 2. 2. 2. CD23 CD Mouse splenocytes: Depletion of non-MZ and non-FO Bcells non-FO and non-MZ of Depletion splenocytes: Mouse Positive selection of FOBcells of selection Positive fraction): (flow-through FOBcells and MZ Pre-enriched Isolation of MZ and FO B cells from mouse for for spleen mouse from Bcells FO and of MZ Isolation Background information Background phenotyping and functional characterization. functional and phenotyping 23 autoMACS Column (program “Depletes”). (program Column autoMACS and Anti-Biotin MicroBeads and Anti-Biotin with Bcells transitional Indirect Magnetic separation using an LS Column or an an or Column LS an using separation Magnetic Direct autoMACS Column (program “Possels”). Magnetic separation using an LS Column or an an or Column LS an using separation Magnetic hi low/– FOB MZ B cells (flow-through fraction) (flow-through Bcells MZ magnetic labeling of FO B cells with with FO Bcells of labeling magnetic magnetic labeling of non-B cells, B1 cells, and B1 and cells, cells, non-B of labeling magnetic cells (eluted fraction) MZ & FO B Cell & FO BCell MZ . Order no. 130-100-366 Biotin-Antibody Cocktail Cocktail Biotin-Antibody CD23

MicroBeads.

140-004-047.02 1.4 ▲ ● ● ● ● ● ● ● are for research useonlyandnotfor diagnostic ortherapeutic use. Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective respective the to refer details For Separators. II SuperMACS™ or VarioMACS™ (Optional) Pre-Separation Filters (30 µm, # (30 µm, Filters Pre-Separation (Optional) (# Kit Removal Cell Dead (Optional) (# Solution Iodide Propidium (Optional) (# mouse 575) 130 092 Reagent, to FcR Blocking (Optional) for flow antibodies Fluorochrome-conjugated (Optional) ▲ of non-MZ Depletion Separators: MACS and Columns MACS phosphate-buffered containing solution a Prepare Buffer: Reagent and instrument requirements instrument and Reagent (# 222). Keep buffer cold 222).(2−8 buffer Keep 2 7-AAD for flow cytometric exclusion of dead cells. dead of exclusion cytometric 7-AAD for flow can be replaced by other proteins such as mouse as such proteins other by replaced be can (CPD). BSA dextrose phosphate (ACD-A) citrate or formula-A dextrose citrate or fetal bovine serum (FBS). Buffers or media containing Ca2 containing media or Buffers (FBS). serum bovine fetal or depletion of cells. dead cytometric analysis, e.g., CD45R (B220)-VioBlue, CD45R e.g., mouse analysis, cytometric or the Pro autoMACS the by using performed be also can remove cell clumps. remove cell recommended for use. for recommended subsequent positive selection of CD23 selection positive subsequent (PBS), pH saline avoid Fc receptor–mediated antibody labeling. labeling. antibody avoid Fc receptor–mediated www.miltenyibiotec.com/antibodies. to refer antibodies about (130-097-819). mouse CD23-PE, and For more information Separator. autoMACS Column. LS on an performed be can non-FO B cells and MACS Separator data sheet. data Separator MACS 091-376) 1:20 with autoMACS® Rinsing Solution (# Solution 091-376) Rinsing autoMACS® with 1:20 performed on an LS Column LS on an performed bubbles could block the column. the block could bubbles autoMACS Column LS Depletion orpositive selection Note: mM EDTA by diluting MACS BSA Stock Solution (# Solution Stock BSA MACS EDTA by diluting mM Note: 130-094-287), CD21/CD35-PE-Vio770,130-094-287), (130-097-216), mouse EDTA can be replaced by other supplements such as anticoagulant anticoagulant as such supplements other by replaced be can EDTA

Column adapters are required to insert certain columns into the the into columns certain insert to required are adapters Column 10⁸ a. number Max. 2×10⁸ of labeledcells 7.2, 0.5% bovine serum albumin (BSA), and albumin serum 7.2, bovine 0.5% Max. number number Max. 4 ×10⁹ 2 ×10⁹ of total cells °C). Degas buffer before use, as air air as use, before buffer °C). Degas . Positive selection and depletion depletion and selection . Positive

serum albumin, mouse albumin, serum 130-090-101) for the + iiAS QuadroMACS, MidiMACS, FO B cells can be be can Bcells FO autoMACS Pro, autoMACS VarioMACS, SuperMACS II Separator 130-093-233) or 130-041-407) to to 130-041-407) + or Mg2 or 130-091- + are not not are

serum, serum, The The 130-

▲ ▲ ▲ ▲ ▲ 10⁸ total cells. When working with fewer than 10⁸ cells, use the same same the use 10⁸ cells, than fewer with working When cells. 10⁸ total 10. 1. 4. When working with lymphoid organs, non organs, lymphoid with working When Working on ice may require increased incubation times. Higher Higher times. incubation increased require may on ice Working 2. To remove dead cells, we recommend using density gradient gradient density using we recommend To cells, remove dead 3.

2.1 2. 7. 9. 6. 5. volumes and total volumes). total and volumes numbers, cell higher with working When indicated. as volumes will prevent capping of antibodies on the cell surface and non- and surface cell on the of antibodies capping prevent will For details see the protocols section at www.miltenyibiotec.com/ section protocols the see For details page 2/5 page centrifugation or the Dead Cell Removal Kit (# Kit 130-090-101). Removal Cell Dead or the centrifugation remove cell clumps which may clog the column. Moisten filter with with filter Moisten column. the clog may which clumps remove cell 8. nylon mesh (Pre-Separation Filters, 30 µm, # µm, 30 Filters, (Pre-Separation mesh nylon for 2×10⁸ total cells, use twice the volume of all indicated reagent reagent indicated of all volume the twice use cells, for 2×10⁸ total gentleMACS the or methods suspension before magnetic labeling. Pass cells through 30 µm µm 30 through cells Pass labeling. magnetic before suspension (e.g. accordingly volumes total and volumes reagent up all scale labeling. cell specific specific cell labeling. cell specific protocols. manual using suspension asingle-cell prepare , peripheral temperatures and/or longer incubation times may lead to non- to lead may times incubation longer and/or temperatures buffer before use. before buffer

For optimal performance it is important to obtain asingle obtain to important it is performance For optimal for up to are below given labeling Volumes for magnetic This solutions. pre-cooled use and cold, cells keep Work fast, MicroBeads. MACS to non-specifically bind may cells Dead 2–8 is temperature incubation recommended The 10⁸ total cells. cells. 10⁸ total Resuspend up to 10⁸ cells in 500 µL of buffer. µL 500 in up10⁸ to cells Resuspend Wash cells by adding 10 by adding Wash cells ▲ Protocol µL of buffer per 10⁸ total cells. cells. per total 10⁸ of buffer µL 400 in pellet cell Resuspend for 10 at 300×g suspension cell Centrifuge Mix well and incubate for 5 incubate and well Mix Add 100 Add 300 Proceed to magnetic separation (2.3). separation magnetic to Proceed 10 additional for an incubate and well Mix number. cell Determine Sample preparation Sample (2−8 MicroBeads per 10 per MicroBeads completely. for 10 at 300×g centrifuge refrigerator (2−8 refrigerator supernatant completely. supernatant Note: °C). °C).

For higher cell numbers, scale up buffer volume accordingly. accordingly. volume buffer up scale numbers, cell higher For 2.2 µL of µL µL of cold buffer and buffer of cold µL cells Magnetic labeling labeling Magnetic MZ & FO B Cell Biotin-Antibody Cocktail Biotin-Antibody BCell &FO MZ °C).] ⁸ total cells. cells. total ™ Dissociator. mL of buffer per 10⁸ cells and cells per 10⁸ of buffer mL minutes. Aspirate supernatant supernatant Aspirate minutes. of non-MZ and non-FO B and of non-MZ minutes in the refrigerator refrigerator the in minutes 200 ‑ lymphoid tissues, or or tissues, lymphoid µL of Anti-Biotin of Anti-Biotin µL minutes. Aspirate Aspirate minutes. 130-041-407) to to 130-041-407) minutes in the the in minutes Order no. 130-100-366 no. Order ‑ per per cell cell °C. °C.

140-004-047.02 ▲ ▲ ▲ ▲ ▲ 1. 4. 3.

2. 6. 5. are for research useonlyandnotfor diagnostic ortherapeutic use. FO B FO cells. For details refer to the section describing the cell separation separation cell the describing section the to refer For details cells. labeled of magnetically frequency the and labeling, of magnetic Depletion with the autoMACS® Pro Separator Separator Pro autoMACS® the with Depletion Columns LS with Depletion or the autoMACS® Separator autoMACS® the or use the autoMACS® Pro Separator or the autoMACS Separator. autoMACS or the Separator Pro autoMACS® the use autoMACS Separator should have a temperature of ≥10 °C. have atemperature should Separator autoMACS of number the and cells of total number the to according programs in the respective user manual. manual. user respective the in programs step. next the to proceeding Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

Program choice depends on the isolation strategy, the strength strength the strategy, isolation on the depends choice Program the or Separator Pro the autoMACS for operating used Buffers on how to for instructions manual user respective the to Refer before empty is reservoir column the until wait Always Separator MACS and Column MACS appropriate an Choose Depletion: Depletion: 5. 4. 3. 2. 1. Proceed to 2.4 for the labeling of CD21 labeling for the 2.4 to Proceed column wash and through pass that cells unlabeled Collect of buffer. 3mL with by rinsing column Prepare Apply cell suspension onto the column. onto the suspension Apply cell Place LS Column in the magnetic field of a suitable MACS suitable of a field magnetic the in Column LS Place (Optional) Remove column from the separator and place place and separator the from column Remove (Optional) Magnetic separation with the autoMACS® Pro Separator Pro autoMACS® the with separation Magnetic with 1×3 mL of buffer. Collect total effluent; this is the is this effluent; total Collect of buffer. 1×3 mL with non-MZ and non-FO B cells and all other non-B cells by firmly by firmly non-B cells other all and non-FO Bcells and non-MZ fraction. Bcell FO and MZ pre-enriched unlabeled Separator. For details see LS Column data sheet. data Column LS see For details Separator. pushing the plunger into the column. the into plunger the pushing labeled magnetically the out flush Immediately column. the it on a suitable collection tube. Pipette 5 mL of buffer onto of buffer 5 mL Pipette tube. collection it on a suitable cells. For details see table in section 1.4. section in table see For details cells. (Optional) Collect positive fraction from row C of the tube tube row Cof the from fraction positive Collect (Optional) of CD21 labeling for the 2.4 to Proceed is This rack. tube row Bof the in fraction negative Collect program: following the choose separation For astandard containing Apply tube instrument. the prime and Prepare collection tubes in rows B and C. Band rows in tubes collection Place fractions. cell unlabeled and labeled the collecting rack. This fraction represents the magnetically labeled labeled magnetically the represents fraction This rack. non-MZ and non-FO B cells and all other non-B cells. other all and non-FO Bcells and non-MZ sample tube in row A of the tube rack and the fraction fraction the and rack tube row Aof the in tube sample the unlabeled pre-enriched MZ and FO B cell fraction. Bcell FO and MZ pre-enriched unlabeled the 2.3 non-MZ and non-FO Bcells and non-MZ Magnetic separation: Depletion of Depletion separation: Magnetic Depletes

the sample and provide tubes for tubes provide and sample the int CD23 int

CD23 hi FO Bcells. FO hi FO Bcells. FO MZ and and MZ

▲ 1. 1. 4. 4. 3. 3.

2. 2. 7. 6. 6. 5. 5. page 3/5 page Positive selection with LS Columns LS with selection Positive numbers, scale up all volumes accordingly. accordingly. volumes up all scale numbers, starting cell number of up to 10⁸ total cells. For higher initial cell cell initial For higher cells. of up10⁸ to total number cell starting

Volumes for magnetic labeling given below are for an initial initial for an are below given labeling Volumes for magnetic Depletion: Depletion: 1. ▲ 5. 4. 3. 2. µL of CD23 MicroBeads of CD23 µL Add 50 of buffer. µL 450 in pellet cell Resuspend Apply cell suspension onto the column and collect flow- collect and column onto the suspension Apply cell of buffer. 3mL with by rinsing column Prepare Remove column from the separator and place it on a suitable it on asuitable place and separator the from column Remove that cells unlabeled Collect of buffer. 3 mL with Wash column 10 by adding Wash cells for 10 incubate and well Mix Pipette 5 mL of buffer onto the column. Immediately flush out flush Immediately column. the onto of buffer 5mL Pipette of buffer. µL 500 in up10⁸ to cells Resuspend for 10 at 300×g suspension cell Centrifuge Place LS Column in the magnetic field of a suitable MACS suitable of a field magnetic the in Column LS Place (2.5). separation magnetic to Proceed Magnetic separation with the autoMACS® Separator autoMACS® the with separation Magnetic (2−8 collection tube. fraction contains the unlabeled enriched MZ Bcells. MZ enriched unlabeled the contains fraction completely. supernatant Aspirate for 10 minutes. supernatant completely. supernatant Separator. For details refer to LS Column data sheet. data Column LS to refer For details Separator. the magnetically labeled cells by firmly pushing the plunger the plunger pushing by firmly cells labeled magnetically the This 3. step from effluent the with combine and through pass through. into the column. This is the enriched FO B cell fraction. cell FO B enriched the is This column. the into Note: This fraction represents the magnetically labeled non-MZ non-MZ labeled magnetically the represents fraction This Prepare and prime the instrument. the prime and Prepare (Optional) Collect positive fraction from outlet port pos1. port outlet from fraction positive Collect (Optional) of CD21 labeling for the 2.4 to Proceed the is neg1. This port outlet from fraction negative Collect program: following the choose separation For astandard containing Apply tube collecting the labeled and unlabeled cell fractions. Place Place fractions. cell unlabeled and labeled the collecting unlabeled pre-enriched MZ and FO B cell fraction. Bcell FO and MZ pre-enriched unlabeled sample tube at the uptake port and the fraction collection collection fraction the and port uptake at the tube sample and non-FO B cells and all other non-B cells. other all and non-FO Bcells and tubes at port neg1 and port pos1. port neg1 and at port tubes °C). °C). 2.5

For higher cell numbers, scale up buffer volume accordingly. accordingly. volume buffer up scale numbers, cell higher For 2.4 Magnetic separation: Positive selection of selection Positive separation: Magnetic CD21 Magnetic labeling of labeling Magnetic Depletes int CD23 hi mL of buffer and centrifuge at 300×g 300×g at centrifuge and of buffer mL FO Bcells FO

the sample and provide tubes for tubes provide and sample the . minutes in the refrigerator refrigerator the in minutes CD21 int CD23 int minutes. Aspirate Aspirate minutes. CD23 Order no. 130-100-366 no. Order hi FO Bcells FO hi FO Bcells. FO

140-004-047.02 3.

CD93 (AA4.1)-APC and analyzed by flow cytometry using the using cytometry by flow (AA4.1)-APCCD93 analyzed and stained fluorescently were cells The mouse. Kit, Isolation Cell fluorescence. are for research useonlyandnotfor diagnostic ortherapeutic use. MACSQuant® Analyzer. Cell debris and dead cells were excluded were excluded cells dead and debris Cell Analyzer. MACSQuant® of 6-week- spleens the from were isolated Bcells FO and MZ with CD45R (B220)-VioBlue CD45R (#with PE-Vio770 (# old BALB/c mice by using the Marginal Zone and Follicular B Follicular and Zone Marginal the by using old BALB/c mice or the Separator Pro autoMACS® the with selection Positive from the analysis based on scatter signals and propidium iodide iodide propidium and signals on scatter based analysis the from autoMACS® Separator autoMACS® Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

CD45R (B220)-VioBlue 1. 3. 2. 3. 2. 1. Example of a separation using the Marginal Marginal the using of aseparation Example Magnetic separation with the autoMACS® Separator autoMACS® the with separation Magnetic Separator Pro autoMACS® the with separation Magnetic Zone and Follicular B Follicular and Zone Prepare and prime the instrument. the prime and Prepare Collect positive fraction from outlet port pos1. This is the is pos1. This port outlet from fraction positive Collect tube neg1 of the port outlet in fraction negative Collect Possels selection: Positive program: following the choose separation For astandard containing Apply tube is This rack. tube row Cof the in fraction positive Collect This rack. tube row Bof the in fraction negative Collect selection: Positive program: following the choose separation For astandard containing Apply tube instrument. the prime and Prepare enriched FO B cell fraction. Bcell FO enriched cells. Place fractions. cell unlabeled and labeled the collecting C. Band rows in tubes collection Place fractions. cell unlabeled and labeled the collecting rack. This fraction contains the unlabeled enriched MZ B enriched unlabeled the contains fraction This rack. fraction contains the unlabeled enriched MZ Bcells. MZ enriched unlabeled the contains fraction sample tube at the uptake port and the fraction collection collection fraction the and port uptake at the tube sample fraction the and rack tube row Aof the in tube sample tubes at port neg1 and port pos1. port neg1 and at port tubes fraction. Bcell FO enriched the CD93-APC 130-097-216), (# CD23-PE Before separation Before Possels

the sample and provide tubes for tubes provide and sample the for tubes provide and sample the Cell Isolation Kit Cell 130-094-287), CD21/CD35- 130-094-287), CD21/CD35-PE-Cy7 130-097-819), and CD23-PE

References4. 1. Warnings deposits in plumbing where explosive conditions may develop. may conditions explosive where plumbing in deposits Miltenyi Biotec provides technical support worldwide. Visit worldwide. support technical provides Biotec Miltenyi www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec Biotec Miltenyi nearest your find to www.miltenyibiotec.com/local 2. Refer to to Refer page 4/5 page contact. running water before discarding. These precautions are recommended to avoid avoid to recommended are precautions These discarding. before water running Reagents contain sodium azide. Under acidic conditions sodium azide yields yields azide sodium conditions acidic Under azide. sodium contain Reagents hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with with diluted be should compounds Azide toxic. extremely is which acid, hydrazoic

CD45R (B220)-VioBlue CD45R (B220)-VioBlue Turchinovich, G. Turchinovich, White, H. N. N. H. White, sequences into the murine CD21 murine the into sequences that seen in the class-switched antibody response. J. Immunol. 188: 287–293. 188: Immunol. J. response. antibody class-switched the in seen that basic Krüppel-like factor (BKLF/KLF3). Blood 117: Blood 3780–3792. (BKLF/KLF3). factor Krüppel-like basic Flow-through fraction: unlabeled enriched MZ Bcells MZ enriched unlabeled fraction: Flow-through www.miltenyibiotec.com CD93-APC CD93-APC et al. et Eluted fraction: enriched FO Bcells FO enriched fraction: Eluted et al. et (2012) Recruitment of a distinct but related set of VH VH of set related but a distinct of (2012) Recruitment (2011) Programming of marginal zone B-cell fate by by fate B-cell zone marginal of (2011) Programming

hi /CD23 for all data sheets and protocols. protocols. and sheets data for all – marginal zone B cell repertoire to to repertoire Bcell zone marginal CD21/CD35-PE-Cy7 CD21/CD35-PE-Cy7 CD23-PE CD23-PE Order no. 130-100-366 no. Order

140-004-047.02 AGREES TO BE BOUND BY THESE TERMS. BY THESE BOUND BE TO AGREES warranty no provides Biotec Miltenyi PLACE. IN LICENSES APPROPRIATE ALL Please notice. prior without change to subject are specifications and information All METHODS. APPLICATION Legal notices Legal contact Miltenyi Biotec Technical Support or visit www.miltenyibiotec.com for the the for www.miltenyibiotec.com visit or Support Technical Biotec Miltenyi contact Trademarks Technical information autoMACS, gentleMACS, MACS, and MACSQuant, MidiMACS, MACSQuant, and MACS, gentleMACS, autoMACS, visit or representative Biotec Miltenyi local your contact Please conditions. and terms separate by excluded be may uses Certain limitations. certain have may and/or as www.miltenyibiotec.com, at website Biotec’s Miltenyi on available Services and applicable product’s the of date expiration the on ending and distributor authorized are for research useonlyandnotfor diagnostic ortherapeutic use. Licenses warranty product Limited worldwide. worldwide. such of completeness or accuracy the but reliable, be to believes Biotec Miltenyi which order, of time the at product the for specifications published Biotec’s Miltenyi with (whether the customer is an academic or for-profit entity). This product may not be be not may product This entity). for-profit or academic an is customer the (whether The purchase of this product conveys to the customer the non-transferable right to to right non-transferable the customer the to conveys product this of purchase The patents issued or pending more or one by covered be may use its and/or product This by provided statements other and protocols, data, information, technical The A IF DETERMINING FOR RESPONSIBLE SOLELY IS CUSTOMER THE TERMS. most up-to-date information on Miltenyi Biotec products. Biotec Miltenyi on information up-to-date most shelf life stated on the product label, packaging or documentation (as applicable) or, (as applicable) documentation or packaging label, product the on stated life shelf persons with knowledge and technical skills sufficient to assess and apply their own own their apply and assess to sufficient skills technical and knowledge with persons rights owned by a third party. BY USE OF THIS PRODUCT, THE CUSTOMER CUSTOMER PRODUCT, THE OFTHIS BY USE party. athird by owned rights BY THESE BOUND BE TO AGREES CUSTOMER PRODUCT, THE OFTHIS USE use the purchased amount of the product in research conducted by the customer customer the by conducted research in product the of amount purchased the use documentation, applicable its with accordance in conditions and use normal under forth in Miltenyi Biotec’s General Terms and Conditions for the Sale of Products Products of Sale the for Conditions and Terms General Biotec’s Miltenyi in forth its or Biotec Miltenyi by product the of delivery of date the on beginning aperiod for substantially conform to and materials and workmanship in defects material from further sold. Additional terms and conditions (including the terms of a Limited Use Use aLimited of terms the (including conditions and terms Additional sold. further Miltenyi Biotec’s website at www.miltenyibiotec.com for more information. more for www.miltenyibiotec.com at website Biotec’s Miltenyi experience or tests, information, on based are document this in Biotec Miltenyi set as terms warranty the to subject provided is Warranty Product Biotec’s Miltenyi free be to product this warrant B.V. affiliate(s) Biotec its KG and/or &Co. Miltenyi Copyright © 2020 Miltenyi Biotec and/or its affiliates. All rights reserved. rights All affiliates. its and/or Biotec Miltenyi ©2020 Copyright ADDITIONAL REQUIRE MAY PRODUCT OFTHIS USE CUSTOMER’S PRODUCT IS SUITABLE FOR CUSTOMER’S PARTICULAR PURPOSE AND AND PURPOSE PARTICULAR CUSTOMER’S FOR SUITABLE IS PRODUCT IS SOLELY RESPONSIBLE FOR DETERMINING FOR ITSELF WHETHER IT HAS HAS IT WHETHER ITSELF FOR DETERMINING FOR RESPONSIBLE SOLELY IS trademarks or trademarks of Miltenyi Biotec and/or its affiliates in various countries countries various in affiliates its and/or Biotec Miltenyi of trademarks or trademarks property intellectual infringe not will and not does product this of use customer’s that herein. contained omissions or errors editorial or technical LICENSES DEPENDING ON THE SPECIFIC APPLICATION. THE CUSTOMER CUSTOMER THE APPLICATION. SPECIFIC THE ON DEPENDING LICENSES apply. may License) Label Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products informed judgment to the information. Miltenyi Biotec shall not be liable for any any for liable be not shall Biotec Miltenyi information. the to judgment informed for intended are data and information technical Such guaranteed. not is information BY apply. may terms Additional Warranty”). (“Product order of time the at effect in Warranty”). (“Product delivery of date from (1) ONE YEAR thereof, absence the in logo, QuadroMACS, SuperMACS, VarioMACS, Vio, and VioBlue and Vio, VarioMACS, SuperMACS, QuadroMACS, the Miltenyi Biotec Biotec Miltenyi the

are registered registered are

page 5/5 page Order no. 130-100-366 no. Order