140-003-597.03 1. Warnings The CD31 reacts with the cell surface CD31. protein The surface cell the with reacts CD31 The antibody CD31 labeled magnetically The in plumbing to avoiddeposits are recommended precautions These 1.2 1.1 1. Description 4. This product is for research use only.use is forresearch product This

Contents 2. Capacity Components 3. Column, which is placed in the magnetic field of a MACS Separator. Separator. MACS of a field magnetic the in placed is which Column, MicroBeads. Then, the cell suspension is loaded onto a MACS® MACS® onto a is loaded suspension cell the Then, MicroBeads. where explosive conditions may develop. may conditions explosive where First, the CD31 the First, Reagents contain sodium azide. Under acidic conditions sodium sodium conditions Under acidic azide. sodium contain Reagents column. The unlabeled cells run through; this cell fraction is fraction cell this through; run cells unlabeled The column. Product format discarding. before water running with diluted be should compounds murine CD31 antigen is also known as PECAM as known also is CD31 antigen murine CD31 retained magnetically the field, magnetic macsde www.miltenyibiotec.com www.miltenyibiotec.com as the positively selected cell fraction. fraction. cell selected positively the as Azide toxic. extremely is which acid, hydrazoic yields azide Storage thus depleted of CD31 depleted thus Phone Phone Friedrich-Ebert-Straße Friedrich-Ebert-Straße Miltenyi Biotec B.V. KG &Co. Biotec Miltenyi

Protocol

References Example of a separation using the CD31 the MicroBeads using of aseparation Example Background information Background Separation MACS® of the Principle 2.3 2.2 2.1 1.4 Applications 1.3 1.2 1.1 Description +49 2204 8306-0, 2204 +49 @

miltenyi.com

Magnetic separation Magnetic labeling Magnetic preparation Sample requirements instrument and Reagent information Background Separation MACS® of the Principle

+ cells are magnetically labeled with CD31 with labeled magnetically are cells For 2×10⁹ total cells, 2×10⁹For total Store protected from at 2−8 light from Store protected 2 CD31 MicroBeads are supplied in buffer buffer in supplied are CD31 MicroBeads vial label. vial MicroBeads conjugated to monoclonal monoclonal to conjugated MicroBeads IgG2a). 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. anti‑ 68, 5142968, Fax Fax mL CD31 MicroBeads, CD31mL MicroBeads, + cells. After removing the column from the from column the removing After cells. +49 2204 85197 2204 +49 mouse CD31mouse Bergisch Gladbach, Germany Bergisch Gladbach, + cells are retained within the the within retained are cells antibodies up to 200 separations. 200 to up + cells can be eluted eluted be can cells - 1. The encoded 1.encoded The (isotype: rat rat (isotype: °C. Do not Do °C. mouse :

1.4 Applications 1.3 mouse CD31 MicroBeads (TEM) under most inflammatory conditions.³ inflammatory most under (TEM) page 1/4 ● ● ● cells and to different degrees on most leukocyte sub leukocyte on most degrees different to and cells a molecule.¹ CD31 is present on mature endothelial endothelial CD31 molecule.¹ on mature present is adhesion a cell the protein is required for leukocyte transendothelial migration migration transendothelial for leukocyte required is protein the properties, adhesive exhibiting in function its Besides .² asingle is protein immunoglobulin

▲ ▲ ▲ Always use freshly prepared buffer. Do buffer. prepared freshly use Always Buffer: Prepare a solution containing phosphate containing solution a Prepare Buffer: CD31 mouse expressing of cells or depletion selection Positive MACS Columns and MACS Separators: CD31 Separators: MACS and Columns MACS Reagent and instrument requirements instrument and Reagent (# (# can be replaced by other such as mouse serum albumin, mouse serum, serum, mouse albumin, serum mouse as such proteins other by replaced be can formula dextrose citrate or fetal bovine serum (FBS). Buffers or media containing Ca containing media or Buffers (FBS). serum bovine fetal or data sheet. Running Buffer or MACSQuant® Running Buffer as they they as Buffer Running MACSQuant® or Buffer Running can also be depleted using MS or LS Columns. Positive Positive Columns. or LS MS using depleted be also can CD31 the antigen express strongly that Cells Columns. of LD use the with or depleted Columns or LS MS by using enriched the affect could that azide of sodium amount a small contain results. recommended for use. for recommended saline (PBS), pH saline selection or depletion can also be performed by using the the by using performed be also can or depletion selection and 2 and antigen. autoMACS Pro or the autoMACS Separator. autoMACS or the Pro autoMACS for optimal recovery during enrichment. during recovery optimal for column. the block could bubbles air as use, before SuperMACS™ II Separators. For details refer to the respective MACS Separator Separator MACS respective the to refer details For Separators. II SuperMACS™ autoMACS Column LD LS MS Positive or depletion selection Depletion Positive selection

130‑091‑222).cold (2−8 buffer Keep Solution Rinsing 130‑091‑376) autoMACS® with 1:20

Note: Note: Note: Note: Note: mM EDTA by diluting MACS BSA Stock Solution Solution Stock BSA MACS EDTA by diluting mM EDTA can be replaced by other supplements such as anticoagulant anticoagulant as such supplements other by replaced be can EDTA For weak expressing cells the use of an LS Column is recommended recommended is Column LS an of use the cells expressing weak For Column adapters are required to insert certain columns into into columns certain insert to required are adapters Column

10 Max. number number Max. of labeledcells 2 1.5 5

- ×

× like (Ig like ⁷ - 10

× 10 pass type I containing six six containing protein Imembrane type pass 10 ⁷ ⁷ ⁷ 7.2, 0.5% bovine serum albumin (BSA), albumin serum 7.2, bovine 0.5% - A (ACD - like) C2 like) 4 2 of total cells - 10 Max. number 3 A) or citrate phosphate dextrose (CPD). BSA dextrose phosphate A) citrate or

×

× × ⁸ 10 10 10 - ⁷ ⁷ typ domains and functions as as functions and domains typ ⁷ auto Super Super Super Separator Midi Midi MiniMACS, OctoMACS, Order no. 130-097-418Order not use MACS MACS MACS °C). Degas buffer buffer °C). Degas MACS MACS MACS Pro, auto , Quadro , Quadro 2+ +

cells can be be can cells II II II or Mg or autoMACS® autoMACS® - types and and types - buffered buffered

2+ MACS MACS MACS are not not are , , 140-003-597.03 ▲ ▲ ▲ ▲ ▲ ▲ 10⁷ total cells. When working with fewer than 10⁷ cells, use the same same the use 10⁷ cells, than fewer with working When cells. 10⁷ total Therefore, these cells have to be depleted, e.g., by using CD45 CD45 using by e.g., depleted, be haveto cells these Therefore, When working with primary tissue prepare asingle prepare tissue primary with working When or tissues, non‑lymphoid organs, lymphoid with working When 2. To remove dead cells, we recommend using density gradient gradient density using we recommend To cells, remove dead 2.1 ● ● ● ● ● volumes and total volumes). total and volumes numbers, cell higher with working When indicated. as volumes are for research useonlyandnotfor diagnostic ortherapeutic use. MicroBeads, mouse (# mouse MicroBeads, For details refer to the protocols section at www.miltenyibiotec.com/ section protocols the to refer For details cell labeling. labeling. cell (# Kit 130 Removal Cell Dead or the centrifugation tissue. primary dissociated from isolated be should cells remove cell clumps which may clog the column. Moisten filter with with filter Moisten column. the clog may which clumps remove cell www.gentlemacs.com/protocols. to refer using manual methods or the gentleMACS Dissociator. For details For details Dissociator. gentleMACS or the methods manual using nylon mesh (Pre mesh nylon for 2×10⁷ total cells, use twice the volume of all indicated reagent reagent indicated of all volume the twice use for 2×10⁷ cells, total Dissociator. gentleMACS or the methods specific cell labeling. Working on ice may require increased increased may require on ice Working labeling. cell specific 30 through cells Pass labeling. magnetic before suspension (e.g. accordingly volumes total and volumes reagent up all scale at different degrees on most leukocyte subtypes and platelets. and platelets. subtypes leukocyte on most degrees at different temperatures and/or longer incubation times may lead to non to lead may times incubation longer and/or temperatures non and surface cell on the of antibodies capping prevent protocols. asingle prepare blood, peripheral incubation times. times. incubation Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products buffer before use. before buffer

For optimal performance it is important to obtain a single‑cell asingle‑cell obtain to important it is performance For optimal for up to are below given labeling Volumes for magnetic pre use and cold, cells keep Work fast, non bind may cells Dead CD31 expressed is cells, on endothelial presence its Besides 2–8 is temperature incubation recommended The ▲ (Optional) gentleMACS™ Dissociator (# Dissociator gentleMACS™ (Optional) Pre (Optional) (# Kit Removal Cell Dead (Optional) (# Solution Iodide Propidium (Optional) Fluorochrome (Optional) Protocol Sample preparation Sample 7 depletion of cells. dead cytometric analysis, e.g., CD31 e.g., analysis, cytometric remove cell clumps. remove cell gentleMACS Octo Dissociator with Heaters (# Heaters with Dissociator Octo gentleMACS (# Dissociator Octo gentleMACS for tissue dissociation when working with primary tissue. primary with working when dissociation for tissue antibodies refer to www.miltenyibiotec.com/antibodies. to refer antibodies be used during tissue dissociation or detachment of cultured cells. cultured of detachment or dissociation tissue during used be -

AAD for flow cytometric exclusion of dead cells. dead of exclusion cytometric for flow AAD

Note: Note: As the CD31 epitope is degraded by trypsin, this enzyme should not not should enzyme this trypsin, by degraded is CD31 epitope the As 2.2 Magnetic labeling Magnetic - Separation Filters, 30 µm, # µm, 30 Filters, Separation - Separation Filters, 30 µm (# µm 30 Filters, Separation 130 - 052 - - conjugated CD31 antibodies for flow CD31conjugated antibodies specifically to MACS MicroBeads. MicroBeads. MACS to specifically - 301), beforehand when endothelial 301), endothelial when beforehand - - PE cell suspension using manual manual using suspension cell . For more information about about . For more information - cooled solutions. This will will This solutions. cooled 130 130 - 090 130 130 130 - - - cell suspension suspension cell 095 090 130 130 - - - - 101) for the 093 041 041 - °C. Higher Higher °C. - 937), or 101) - - 096 093 - - - - 233) or 407) to to 407) specific specific 407) to to 407) . - - 235), 427) 427) µm µm

-

▲ ▲ 1. 1. 4. 4. 3. 3. 2. 2. 7. 7. 9. 6. 6. 5. 5. For details refer to the table in section 1.4. section in table the to refer For details page 2/4 page Magnetic separation with MS or LS Columns or LS MS with separation Magnetic 8. according to the number of total cells and the number of CD31 number the and cells of total number the to according proceeding to the next step. next the to proceeding

Always wait until the column reservoir is empty before before empty is reservoir column the until wait Always Separator MACS and Column MACS appropriate an Choose ▲ µL of CD31 MicroBeads per 10⁷ per ofAdd CD31 10 µL MicroBeads μL Add 90 Apply cell suspension onto the column. Collect flow Collect column. onto the suspension Apply cell Wash column with the appropriate amount of buffer. Collect Collect of buffer. amount appropriate the with Wash column Wash cells by adding 1−2 by adding Wash cells (Optional) Add staining antibodies, e.g., 10 e.g., antibodies, Add staining (Optional) (Optional) To increase the purity of CD31 purity the To(Optional) increase ▲ ▲ Determine cell number. number. cell Determine Centrifuge cell suspension at 300×g for 10 at 300×g suspension cell Centrifuge Mix well and incubate for 15 incubate and well Mix Pipette the appropriate amount of buffer onto the column. column. the onto of buffer amount appropriate the Pipette of amount appropriate the with by rinsing column Prepare MACS suitable of a field magnetic the in column Place Proceed to magnetic separation (2.3). separation magnetic to Proceed Remove column from the separator and place it on a suitable it on asuitable place and separator the from column Remove Resuspend up to 10⁸ cells in 500 µL of buffer. µL 500 in up 10⁸ to cells Resuspend reservoir is empty. is reservoir

(2−8 (2−8 Repeat the magnetic separation procedure as described in in described as procedure separation magnetic the Repeat cells labeled magnetically the out flush Immediately data sheet. collection tube. cells. unlabeled containing completely. for 10 at 300×g centrifuge unlabeled cells that pass through and combine with the flow the with combine and through pass that cells unlabeled fraction can be enriched over a second MS or LS Column. Column. or LS MS over asecond enriched be can fraction supernatant completely. supernatant steps 1 to 6 by using a new column. anew 6by 1to using steps Column MACS respective the to refer For details Separator. and incubate for 5 incubate and µL of buffer. of µL 500 through from step 3. step from through column. the into the plunger pushing by firmly buffer: Note: Note N ote: °C). °C). °C). 2.3

:

Perfor

For depletion with LD Columns, resuspend up to 1.25×10⁸ cells in in cells 1.25×10⁸ to up resuspend Columns, LD with depletion For For higher cell numbers, scale up buffer volume accordingly. accordingly. volume buffer up scale numbers, cell higher For Magnetic separation Magnetic of buffer per 10⁷ total cells to the cell pellet. cell the to cells per total 10⁷ of buffer m washing steps by adding buffer aliquots only when the column column the when only aliquots buffer adding by steps m washing 1 mL MS: µL 3×500 MS: 5 MS: minutes in the dark in the refrigerator refrigerator the in dark the in minutes µL 00

mL of buffer per 10⁷ cells and per cells 10⁷ of buffer mL minutes. Aspirate supernatant supernatant Aspirate minutes. minutes in the refrigerator refrigerator the in minutes LS: 5 mL LS: 3×3 mL LS: 3 mL LS: total cells. total minutes. Aspirate Aspirate minutes. + cells, the eluted eluted the cells, µL of CD31µL Order no. 130-097-418 no. Order

- through through

+ cells. - PE - ,

140-003-597.03 ▲ ▲ 1. 4. 3.

2. are for research useonlyandnotfor diagnostic ortherapeutic use. Magnetic separation with the autoMACS® Pro Separator Separator Pro autoMACS® the with separation Magnetic Depletion with LD Columns Columns LD 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 Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

Buffers used for operating the autoMACS Pro Separator or the or Separator Pro the autoMACS for operating used Buffers on how to for instructions manual user respective the to Refer Apply cell suspension onto the column. onto the suspension Apply cell 1. 3. 2. 3. 2. 1. Magnetic separation with the autoMACS® Separator autoMACS® the with separation Magnetic Separator Pro autoMACS® the with separation Magnetic Collect unlabeled cells that pass through and wash wash and through pass that cells unlabeled Collect mL of buffer. 2 mL with by rinsing column Prepare MACS suitable of a field magnetic the in Column LD Place column with 2×1 with column steps by adding buffer two times. Only add new buffer when when buffer new add Only times. two buffer by adding steps sheet. data Column LD the to refer For details Separator. the column reservoir is empty. is reservoir column the washing Perform fraction. cell unlabeled the is this Depletion: Depletion: selection: Positive rack. tube row Bof the in fraction negative Collect Deplete_s Depletion: Possel_s selection: Positive Prepare and prime the instrument. the prime and Prepare For a standard separation choose one of the following following one of the choose separation For astandard containing Apply tube rack. tube row Cof the in fraction positive Collect following one of the choose separation For astandard containing Apply tube instrument. the prime and Prepare Collect negative fraction from outlet port neg1. port outlet from fraction negative Collect pos1. outlet from fraction positive Collect collecting the labeled and unlabeled cell fractions. Place Place fractions. cell unlabeled and labeled the collecting C. Band rows in tubes collection Place fractions. cell unlabeled and labeled the collecting 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 programs: pos1. neg1 and at port tubes programs: Deplete_s mL of buffer. Collect total flow total Collect of buffer. mL Possel_s

the sample and provide tubes for tubes provide and sample the for tubes provide and sample the

- through; through;

Analyzer. Cell debris and dead cells were excluded from the analysis analysis the from were excluded cells dead and debris Cell Analyzer. References4. 1. 3. Column, and an OctoMACS™ Separator. Cells were fluorescently were fluorescently Cells Separator. OctoMACS™ an and Column, Mouse CD31 Mouse Miltenyi Biotec provides technical support worldwide. Visit Visit worldwide. support technical provides Biotec Miltenyi www.miltenyibiotec.com www.miltenyibiotec.com 2. Refer to to Refer page 3/4 page 3. mixture of U937 and bEnd.3 cells using CD31 MicroBeads, an MS MS an CD31 using MicroBeads, cells of U937 bEnd.3 and mixture stained with CD31 with stained Support contact information. contact Support based on scatter signals and propidium iodide fluorescence. iodide propidium and signals on scatter based Example of a separation using using of aseparation Example Woodfin, A. Woodfin, van Buul, J.D. and Hordijk, P.L. (2008) Endothelial signalling by Ig by signalling Endothelial P.L. (2008) Hordijk, and J.D. Buul, van Xie, Y. and Muller, W.A. (1993) Molecular cloning and adhesive properties properties adhesive and cloning W.A. (1993) Y. Muller, Molecular and Xie, CD31 MicroBeads adhesion molecules. Transfus. Clin. Biol. 15(1 Biol. Clin. Transfus. molecules. adhesion of murine /endothelial 1. Proc. Natl. Acad. Sci. Sci. Acad. Natl. 1. Proc. molecule adhesion cell platelet/endothelial murine of 2514–2523. U.S.A. 90: 5569–5573. 90: U.S.A. inflammation and vascular biology. Arterioscler. Thromb. Vasc. Biol. 27(12):Biol. Thromb.Vasc. Arterioscler. biology. vascular and inflammation www.miltenyibiotec.com + endothelial cells (bEnd.3) were isolated from a from were isolated (bEnd.3) cells endothelial et al. et

- PE and analyzed using the MACSQuant® MACSQuant® the using analyzed PE and (2007) PECAM (2007) Side scatter Side scatter Side scatter 1 1 1 CD31 CD31 250 500 750 250 500 750 250 500 750 Before separation Before 000 000 000 0 0 0 -1 -1 -1 for local Miltenyi Biotec Technical Technical Biotec Miltenyi for local + – cells cells 0 0 0 1 1 1 CD31-PE CD31-PE CD31-PE for all data sheets and protocols. protocols. and sheets data for all 10 10 10 - 1: amulti ¹1 ¹1 ¹1 - 0² 0² 0² 2): 3–6. the 10 10 10 - functional molecule in in molecule functional

³ ³ ³

Order no. 130-097-418 no. Order - like cell cell like

140-003-597.03 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. 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