CD43 Microbeads Are Developed for Isolation the of Untouched CD43 First CD43 the Product Format Expressed on I.E

140-001-138.04 1. Description Then, theThen, cell suspension is loaded ontocolumn a which is in placed 1.1 1. Description 1.2 4.

2. Index Components 3. CD43 MicroBeads are developed for isolation the of untouched CD43 First CD43 the Product format expressed on i.e. most leukocytes, Tcells, NKcells, granulocytes, B cells. regulated during transition to pre-B the stage. cell retained CD43 removal of column the from magnetic the field,the magnetically glycoprotein that is suggested involved to be in adhesion, anti- monocyte and macrophages, hematopoietic stem cells and platelets fraction. macsde www.miltenyibiotec.com www.miltenyibiotec.com activated Bcells and plasma cells but not on resting Bcells, e.g. naive adhesion, and transduction signal processes. Size Storage human on magnetic the B cells depletion based of cells that express the CD43antigen.the sialophorin) CD43(leukosalin, is surface acell through and is depleted fraction cell this of CD43 magneticthe field of aMACS®Separator. Thelabeled magnetically but not on erythrocytes. Phone Phone Friedrich-Ebert-Straße Friedrich-Ebert-Straße Miltenyi Biotec B.V. KG &Co. Biotec Miltenyi References Protocol

Example ofExample aseparation using CD43MicroBeads Principle of MACS Background and productapplications 2.3 2.2 2.1 1.3 1.2 1.1 + +49 2204 8306-0, 2204 +49 @ cells are retained on column. the run cells The unlabeled 2

In bone marrow, CD43is found on pro-B cells but is down miltenyi.com Magnetic separation Magnetic labelingMagnetic Sample preparation Reagent and instrument requirements Background and productapplications Principle of MACS

+ + cells are with CD43MicroBeads. labeled magnetically

cells can be eluted cells can be as positively the cell selected The expirationdate is indicatedlabel. thevial on For 10 Store protected from light at not 4−8°C.Do freeze. 2 mLCD43MicroBeads, human: CD43 MicroBeadsare supplied as asuspension (isotype: mouse(isotype: IgG1). MicroBeads conjugated to containing stabilizer monoclonal anti-human CD43antibodies 68, 5142968, Fax Fax ® +49 2204 85197 2204 +49 separation 2-4 9 total cells, up to 100separations. Among B cells, CD43 is expressed on Bergisch Gladbach, Germany Bergisch Gladbach, ® separation

and azide. 0.05%sodium 1 The CD43 antigen is

+ cells. After

"General Protocols""General User inthe Manuals or visit www.miltenyibiotec. 1.3 When working with anticoagulated or peripheralcoat, blood buffy 2.

human CD43 MicroBeads

2.1 ● ● ● ● ● ● ● Examples ofExamples applications PBMC should isolated be by density gradient centrifugation (see com). page 1/3

autoMACS 2×10 10 XS LS 10 MS 10 Column Protocol VarioMACS or SuperMACS. For MACS details, see Separator data sheets. MACS Columns and MACS Separators: ▲ Buffer (degassed): Preparesolution a containing PBS (phosphate ▲ (Optional) Pre-Separation Filter (#130-041-407). (Optional) PI (propidium or iodide) 7-AADfor flowcytometric Isolation of untouched resting Bcells from peripheral blood Positive or selection depletion of cells expressing human the (Optional) Fluorochrome-conjugated antibodies against Reagent and instrument requirements Choose the appropriate the Choose MACS Separator and MACS Columns ▲ Sample preparation calf serum. Buffers serum. calf containing or media Ca dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be MACS BSAStock Solution (#130-091-376)1:20with autoMACS™ CD43 antigen. CD20-PE #130-091-109),CD43and CD235a(Glycophorin A). CD19-PE #130-091-247,CD20-FITC #130-091-108,or Rinsing Solution (#130-091-222).Keep buffercold (4−8°C). cells. or single-cell suspensions from tissue (e.g. lymphoid tissue). exclusion of cells. dead supernatant. Repeat washing step and carefully remove supernatant. mononuclear cells (PBMC), fluids body (e.g. bronchiallavage) according to number the of cells and labeled to number the of total replaced by other proteins such as human albumin, human serum or fetal serum a B cell lineagea Bcell marker (e.g. CD19-APC # 130-091-248, buffered pH7.2,0.5%BSA saline) and 2mMEDTA by diluting in buffer andcentrifuge at 200×g for 10−15 minutes Carefullyat 20°C. remove

Note: Note: EDTA replaced can be by other supplements such as anticoagulant citrate Remove platelets after densitygradient separation of numbermax.

Note:

cells labeled Column adapters are required columns certain to insert into

9 8 7 8 of total cells max. numbermax.

4×10 2×10 2×10 2×10

9 9 8 10

or Mg

Order No. 130-091-333

MiniMACS, OctoMACS, MidiMACS, QuadroMACS, SuperMACS autoMACS Separator VroAS SuperMACS VarioMACS, VarioMACS, SuperMACS 2+ are not recommended for use. : resuspend cell pellet cell resuspend

140-001-138.04 ▲ ▲ ▲ ▲ 1. 1. 4. 4. When working with tissues, prepare a single-cell suspension by a 3. 3. 30 µmnylon mesh (Pre-Separation Filter #130-041-407)to remove ▲ ▲ (see table in section 1.3). table insection (see 2. 2. 7. 9. 6. 5. volumes). are for research useonlyandnotfor diagnostic ortherapeutic use. Manuals or visit www.miltenyibiotec.com). cell labeling.cell cell clumpscell which may clog column. the suspensioncell before magnetic separation. Pass cells through volume the twice cells, use of indicated all reagent volumes and total cells. When working with fewer than 10 reagent volumes and total volumes accordingly (e.g. for 2×10 Magnetic separation with MSor separation with Magnetic LSColumns 8. standard preparation Protocols" "General (see method User inthe according to number the of total cells and number the of CD43 as indicated. When working with numbers, higher cell up all scale prevent capping of antibodies on and surface cell the non-specific Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

Choose an appropriate Choose MACS Column and MACS Separator Volumes for magnetic labeling given below are for up to 10 Work fast, keep cells cold, and pre-cooled use solutions. Thiswill For optimal performance it is important to obtain asingle- Proceed to magnetic separation (2.3). Resuspend up to 10 ▲ Apply suspension cell onto column. the Prepare column by rinsing with appropriate amount of buffer: Mix well and incubate for 15minutes at 4−8°C. Add 20µLof 10 CD43MicroBeads per suspension cell Centrifuge at 300×gfor 10minutes. Pipette off Resuspend cell pellet in80µLofResuspend pellet cell bufferper 10 Determine cell number.Determine cell Place column magnetic inthe field of a suitable MACSSeparator Wash cells by adding 1−2mLof bufferper 10 (Optional) Add staining antibodies, e.g. add 10µLof CD19-PE

Collect unlabeled cells which pass through and which pass through cells unlabeled Collect (see "Column data(see sheets"). (# 130-091-247),and incubate for 5minutes at 4−8°C. dead cells, wedead recommend using density gradient centrifugation (e.g. Ficoll- wash column with appropriatewith column wash amount of buffer. supernatant completely. supernatant at 300×gfor 10minutes. Pipette off supernatantcompletely. Paque™) Removal Kit or Cell Dead the (#130-090-101). temperatures and/or longer incubation to times lead non-specificlabeling. cell Note:

Note: Note: MS: 500µL 2.3

Dead cells may Dead bind non-specifically to MACS MicroBeads. To remove For numbers, higher cell up buffer scale volume accordingly. 2.2 Working on ice Magnetic separation Magnetic Magnetic labelingMagnetic 8 cells in500µLof buffer.

may require increased incubation times. Higher LS: 3mL. 7 cells, use the same the cells, use volumes 7 total cells. 7 cells and centrifuge 7 total cells. 7 7 + total total total total cells cells

"D Column data sheet". ▲ 1. 1. 1. 4. 4. 3. 3. ▲ ▲ 2. 2. 2. 6. 5. For instructions on column the assembly and separation, the refer to For instructions on column assembly and separation, refer to the Magnetic separation with XS Columns XS separation with Magnetic Magnetic separation with the autoMACS the separation with Magnetic Depletion with LD Columns LDColumns with Depletion Depletion with D Columns DColumns with Depletion CSColumns with Depletion use the autoMACS the use the "XS Column data "XS the sheet". page 2/3 page Refer to "autoMACS the Attach a22Gflow resistor the 3-way-stopcockto the of assembled Collect total effluent.Collect fraction. cell unlabeled This the is Depletion: "Deplete" Positive "Possel" selection: Apply suspension cell onto column the Prepare column by rinsing with 2mLof buffer. Place tube containing Apply suspension cell onto column. the Prepare column by filling rinsingand with 60mL of buffer. cells which pass through unlabeled andCollect wash column Remove column from separator the and place it on asuitable Collect unlabeled cells which pass through unlabeled andCollect wash column Pipette appropriate amount of buffer ontothecolumn. Place LDColumn magnetic inthe field of a suitable MACS Prepare and prime autoMACS Separator. Assemble CSColumn and place it magnetic inthe field of a

autoMACS User Manual: "autoMACS Separation Cell Programs". over asecond, freshly prepared column. with 2×1 mL of buffer. total effluent.Collect is This the unlabeled with 30mLbufferfrom the top. total effluent.Collect is This the Performbywashingsteps adding buffer three times, Immediately flush out with magnetically the fraction cell fraction. cell column. collection tube. column the is reservoir empty. once each time column "CSColumn data (see sheet"). magnetic labeling and of frequency the cells. For labeled magnetically details see unlabeled cell fraction. cell unlabeled following separation programs: Separator "LDColumn data (see sheet"). suitable MACS Separator "CSColumn data (see sheet"). autoMACS Separator. For astandard separation, choose labeled cells bylabeled firmly applyingthe plunger suppliedwiththe Note: Note: MS: 3×500µL MS: 1mL To increase purity the it of fraction, passed labeled can magnetically the be

Program choice dependson isolation the strategy, strength the of ™ Separator. ™ User Manual" for instructions on how to

the magnetically labeled cells in the cells inthe labeled magnetically the LS: 5mL. LS: 3×3mL.

™ . Separator

Order No. 130-091-333

140-001-138.04 A)-FITC. Cell debrisA)-FITC. and Cell cells were dead excluded from analysis the 4. 1. Woodman 3. 3.

4. CD19-PE (#130-091-247),CD43-APCand CD235a(Glycophorin are for research useonlyandnotfor diagnostic ortherapeutic use. 2. Barclay 3. Moore Separator with an LS Column. Thecells are fluorescentlystained with Separation of PBMC using CD43MicroBeadsand aMidiMACS Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products based on scatterbased and signals PI fluorescence. References When using program the fraction unlabeled "Deplete", collect When using Example ofExample aseparation using CD43MicroBeads Remold-O‘Donnell (1987)Biochemistry. 26:3908-39134. (outlet "neg1"). port Thisthe CD43 is (outlet Thisthe CD43 "pos1"). is port purified the untouchedthe resting Bcells. London et al. et al. et al. (1994)J. Immunol 153:4978-4987 eds. 1997. The Leucocyte AntigenLeucocyte 1997.The eds. AcademicFactsBook. press. (1998) J Exp Med. (1998)JExp 188:2181-2186

CD235a-FITC CD43-APC CD43-APC programthe "Possel", positive collect fraction CD19 CD19 Before separation CD19-PE CD19-PE CD19-PE + + CD43 CD43 – – B cells B B cells B - cell fraction containing fraction cell + cell fraction. cell

™ APPROPRIATE LICENSES INPLACE. Miltenyi Biotec provides no warranty that Additional terms and conditions (including terms the of aLimited Use License) Label informationAll and specifications are subject to changewithout prior notice. Please Warnings The purchase this of productconveys customer the to the non-transferableright to use This product and/or mayits use coveredbe by one or morepending or issuedpatents The information,technical data, protocols, and other statements provided by Miltenyi Legal notices Legal customer’s of use product this not does andnot will infringe intellectual property rights contact Miltenyi Biotec Technical Support or visit www.miltenyibiotec.com for most the Technical information Trademarks acid, which is extremely toxic. Azide compounds should diluted be with running water autoMACS, MACS, MidiMACS, and conditions. contact Please your Miltenyi local Biotec representative or visit Miltenyi and/or may have limitations. certain may uses excluded be Certain by separate terms distributor and ending on expiration the date of product’s the applicable shelf life stated on Miltenyi Biotec’s website at www.miltenyibiotec.com, as ineffect timethe at of order on product the label, packaging or documentation (as applicable) or, absence inthe owned by athird party. BYUSE OF THISPRODUCT, THECUSTOMER AGREES TO editorial errors or omissions contained herein. 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 Limited product warrantyLimited Licenses where explosive conditions may develop. with knowledge and sufficient technical skills to assess and apply theirown informed (“Product Warranty”). Additional terms may apply. BYUSE OF THISPRODUCT, THE normal and use conditions inaccordance with its applicable documentation, for aperiod Refer to to Refer THE CUSTOMER ISSOLELY RESPONSIBLE FOR DETERMINING IFAPRODUCT contact. mentioned publication inthis are property the of respective their owners and are used may apply. up-to-date information on Miltenyi Biotec products. from material inworkmanship defects and materials and to conform substantially with for identification only. purposes METHODS. Miltenyi Biotec’s published specifications forthe product timethe at of order, under Miltenyi Biotec B.V. &Co. KG and/or its affiliate(s) warrant this be free product to Miltenyi Biotec and/or its affiliates in various countries worldwide. CUSTOMER AGREES TO BYTHESE BE BOUND TERMS. Copyright ©2020Miltenyi Biotec and/or its affiliates. rightsAll reserved. QuadroMACS, SuperMACS, and VarioMACS CUSTOMER’S USE OF THIS PRODUCT MAY REQUIRE ADDITIONAL LICENSES Product Warranty is provided subject to warranty the terms forth as inMiltenyi set Ficoll-Paque is atrademark of GEHealthcare companies IS SUITABLE FOR CUSTOMER’S PARTICULAR PURPOSE ANDAPPLICATION Reagents contain azide. sodium Under acidic conditions azide yields sodium hydrazoic RESPONSIBLE FOR DETERMINING FOR ITSELF WHETHER ITHAS ALL thereof, ONE (1)YEARfrom date of (“Product delivery Warranty”). Miltenyi Biotec’s the customerthe is an academic or for-profit entity). This product sold. may further be not purchasedthe amount of product the inresearch conducted by customer the (whether Biotec’s Terms General and Conditions for of available Sale the Products and Services BE BOUND BYTHESEBE BOUND TERMS. Biotec’s website at www.miltenyibiotec.com for more information. Biotec reliable, to believes be but or completeness accuracy the of such information Biotec document inthis are on information, based tests, or which Miltenyi experience judgment to information. the Miltenyi Biotec not shall liable be for any technical or DEPENDING ON THESPECIFIC APPLICATION. THECUSTOMER ISSOLELY is not guaranteed. Such technical information and data are intended for persons beginning onbeginning date the of of delivery product the by Miltenyi Biotec or its authorized before discarding. These precautions are recommended to avoid deposits in plumbing page 3/3 page www.miltenyibiotec.com

the Miltenyi the Biotec logo, for all data sheets and protocols. protocols. and sheets data for all

are registered trademarks or trademarks of . MiniMACS, OctoMACS, All otherAll trademarks Order No. 130-091-333