140-000-563.09 1. Description The unlabeled The 1.1 1. Description 4/Neuropilin-1) 4.

Contents 2. Capacity Components 3. CD304 CD304 First, CD304 the Product format over asecond column. containing the suspensioncell onto is loaded aMACS® Column which is placed in fraction. fraction. from magnetic the field,the magnetically retained CD304 macsde www.miltenyibiotec.com www.miltenyibiotec.com Storage the magneticthe field of aMACSSeparator. Thelabeled magnetically labeled with CD304 labeled Phone Phone Friedrich-Ebert-Straße Friedrich-Ebert-Straße Miltenyi Biotec B.V. KG &Co. Biotec Miltenyi References Protocol Example ofExample aseparation using CD304(BDCA-4/ the Principle of MACS 2.3 2.2 2.1 1.4 1.3 Applications 1.2 1.1 Neuropilin-1) Kit MicroBead +49 2204 8306-0, 2204 +49 @ (BDCA-4/Neuropilin-1)

(BDCA-4/Neuropilin-1) miltenyi.com

To increase purity, the positively the fraction cell selected Magnetic separation Magnetic labelingMagnetic Sample preparation Reagent and instrument requirements Background information Principle of MACS the cells run through; is cells run thus fraction this depleted of

+ CD304 cells can be eluted cells can be as positively the cell selected The expirationdate is indicatedlabel. thevial on Store protected from light at not 2−8°C.Do freeze. 2 mLFcR Reagent, human: Blocking For 2×10 The products are supplied in buffer containing 2 mLCD304(BDCA-4/Neuropilin-1) Human IgG. Neuropilin-1) antibodies mouse (isotype: IgG1). MicroBeads, human: MicroBeads, stabilizer to monoclonal anti-human CD304(BDCA-4/ (BDCA-4/Neuropilin-1) 68, 5142968, (BDCA-4/Neuropilin-1) Fax Fax (BDCA-4/Neuropilin-1) ® +49 2204 85197 2204 +49 Separation 9

and azide. 0.05%sodium total cells, up to 20separations. Bergisch Gladbach, Germany Bergisch Gladbach, + + cells are retained within column. the cells. After removingthecolumn ® Separation MicroBeads conjugated + MicroBeads. Then, the MicroBeads. Then, cells are magnetically + cells is separated

(BDCA-

Additionally, Neuropilin-1 is known expressed to be on numerous 1) but expression of CD304(BDCA They lack They expression of lineage markers (CD3,CD14,CD16,CD19, 1.2 1.4 1.3 Applications human Neuropilin Kit ‑1) MicroBead CD304 (BDCA-4/

(BDCA-4/Neuropilin-1) on plasmacytoid dendritic cells allows their (BDCA-2) ● ● ● CD304 (BDCA-4/Neuropilin-1) CD33, nor Fc receptors, such as CD32,CD64,or FcεRI. Freshly CD20, CD56),and express neither myeloid markers, like CD13and CD304 (BDCA-4/Neuropilin-1) direct isolation by positive In and selection. blood bone marrow, dendritic cells from peripheral cord blood, or blood, bone marrow, Isolation of CD304(BDCA-4/Neuropilin-1) In contrast to CD304(BDCA-4/Neuropilin-1), binding of antibody characterized as beingCD11c cord and blood¹², bone marrow⁶. Exclusive expression of CD304 cells. on some other cells, primarily follicular Bhelper Tcells. memory expression is, apart from plasmacytoid detected dendritic cells, also cells and monocytes do not express CD304(BDCA-4/Neuropilin- cells.⁵ non-hematopoietic e.g. neurons, types, cell endothelial and tumor for example, plasmacytoid inhuman dendritic cells (PDCs) peripheral blood¹ type Iinterferontype ininfluenzavirus-stimulated plasmacytoid dendritic to CD303 (BDCA-2) shown has been to inhibit production the of in culture.¹ isolated CD1c(BDCA-1) page 1/4

1:20 with autoMACS™ Rinsing Solution (# ▲ Buffer: Preparesolution a containing phosphate-bufferedsaline for studies on dendritic activation,⁴ cell migration,³ cytokine to examine expression of Toll-like receptors,⁴ Background information Reagent and instrument requirements (PBS), pH dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). replaced BSAcan be EDTA by diluting MACS BSA Stock Solution (# could block column. the receptors,³ production,⁴ Buffers containing or media Ca buffercold (2−8 by other proteins such as human albumin, human serum or bovine fetal serum, serum.

Note: +

, CD141(BDCA-3) , EDTA replaced can be by other supplements such as anticoagulant citrate ⁶ In inflamed tonsils, CD304(BDCA , 7.2, 0.5% bovine serum albumin7.2, 0.5%bovine (BSA),and serum 2 ⁸ , ¹⁰ or antigens² new , ⁶ , ⁸ and polarization⁴ Tcell °C). Degas buffer°C). Degas before use, as air bubbles + or CD141(BDCA 2+ or Mg – , CD123 dim + 1 plasmacytoid dendritic cells are 2+ is specifically expressed is specifically by , CD1c(BDCA-1) are not recommended for use. , ⁹ or ⁹ high ‑4/Neuropilin-1) is induced Order no. 130-090-532 , CD4 + , ⁶

, plasmacytoid plasmacytoid ¹³. ‑3) + 130-091-222). Keep , CD45RA ++ ‑4/Neuropilin-1) blood dendritic dendritic blood , ⁷ , ⁸ , – ¹¹ 130-091-376) , and CD2

chemokine chemokine + , CD303 mM – – ⁵, 6 .

140-000-563.09 ▲ When working with tissues or prepare blood, lysed asingle-cell When working with anticoagulated or peripheralcoat, blood buffy 2. ▲ 2.1 Dead Cell Removal Kit Cell Dead (# 130-090-101). ● ● ● ● ●

are for research useonlyandnotfor diagnostic ortherapeutic use. www.miltenyibiotec.com/protocols. Protocols are available also at www.miltenyibiotec.com/protocols. Protocols of respective section the separator manual. user General The For Protocols General the details see of respective section the dead cells, wedead recommend using density gradient centrifugation or the density gradient centrifugation, for example, using Ficoll-Paque™. suspension using standard For methods. General the details see separator manual. user General The Protocols are also available at peripheral blood mononuclearperipheral blood cells (PBMCs) should isolated be by Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

autoMACS Positive selection XS LS MS Positive selection Column Protocol Dead cells mayDead bind non-specifically to MACS MicroBeads. To remove APC (#130-090-905)for identification of plasmacytoid VarioMACS™ or SuperMACS™ Separators. For respective the MACS details see (Optional) Fluorochrome-conjugated antibodies for flow ▲ (Optional) Pre-Separation Filters (# 130-041-407) to remove cell Removal Kit(Optional) Cell Dead (# (Optional) Propidium (PI) iodide or 7-AAD for flowcytometric ▲ MACS Columns and MACS Separators: CD304(BDCA Sample preparation (# CD303 (BDCA-2)-PE (# Columns (positive Positive selection). be can also selection www.miltenyibiotec.com. For more information about other fluorochromeconjugates see depletion of cells. dead dendritic cells; or CD123-FITC (# clumps. exclusion of cells. dead orcytometric fluorescence microscopicevaluation of MACS Neuropilin-1) supernatant. Repeat washing step. separation, e.g. Separator. performed by using autoMACS the or autoMACS the Pro in combination with CD304(BDCA-4/Neuropilin-1) the Kit. MicroBead in buffer andcentrifuge at 200×g for 10−15 Separator data sheet.

Note: Note: Note: 130-090-899), or CD123-APC(#130-090-901)for control.

To remove platelets after densitygradient separation, resuspendpellet cell CD304 (BDCA-4/Neuropilin-1) antibodies are not recommended for staining Column adapters are required columns certain to insert into the cells of labeled Max. number 2 + × 10⁸ 10⁹ 10⁸ 10⁷ cells can be enrichedcells can by be using MS, LS, or XS

CD303 (BDCA-2)-FITC (# Max. number of total cells

130-090-511), or CD303 (BDCA-2)- 2 4 × 10⁹ 2 × 10⁹ 2 × 10⁸ × 10¹⁰

minutes at 20 130-090-897), CD123-PE autoMACS, autoMACS Pro SuperMACS MidiMACS, QuadroMACS, VarioMACS, SuperMACS MiniMACS, OctoMACS, Separator VarioMACS, SuperMACS 130-090-101) for the °C. Carefully aspirate 130-090-510),

‑4/

▲ ▲ ▲ ▲ ▲ ▲ 1. 1. 10. 4. 3. 30 µmnylon mesh (Pre-Separation Filters # ▲ (BDCA-4/Neuropilin-1) 2. 2. 7. 9. 6. 5. volumes). 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 same the 10⁸ cells, use volumes labeling.cell Positive MSor with LSColumns selection reagent volumes and total volumes accordingly (e.g. for 2×10⁸total runs. 8. use. specific cell labeling. cell specific as indicated. When working with numbers, higher cell up all scale according to number the of total cells and number the of CD304 temperatures and/or longer incubation times may to lead non- prevent capping of antibodies on and surface cell the non-specific page 2/4 page Volumes for magnetic labeling given below are for up to 10⁸total To achieve highest purities, perform two consecutive column an appropriate Choose MACS Column and MACS Separator Work fast, keep cells cold, and pre-cooled use solutions. Thiswill Working on icemay require increased incubation times. Higher For optimal performance it is important to obtain asingle- 10⁸ total cells. Resuspend up to 10⁸cells in500µLof buffer. Wash cells by adding 5−10 mL of bufferpercells 10⁸ andcentrifuge Resuspend cell pellet in300µLofResuspend pellet cell bufferper 10⁸ cells.total suspension cell Centrifuge at 300×gfor 10minutes. Aspirate Prepare column by rinsing with appropriate amount of buffer: Add 100µLof FcR Blocking Reagent 10⁸total cells. per Add 100µLof CD304(BDCA-4/Neuropilin-1) MicroBeadsper Mix well and incubate for 15minutes refrigerator inthe number.Determine cell Place column magnetic inthe field of a suitable MACSSeparator. Proceed to magnetic separation (2.3). (Optional) Add staining antibodies after 10 minutes of (2−8 °C). (2−8 °C). 510), and incubate for 5minutes dark inthe refrigerator inthe For respective the MACS details see Column data sheet. supernatant completely. supernatant at 300×g forat 10minutes. 300×g Aspirate supernatant completely. incubation, e.g. add 50µLof CD303(BDCA-2)-FITC (#130-090-

Note: MS: 500µL 2.3

For numbers, highercell up buffer scale volume accordingly. 2.2 Magnetic separation Magnetic Magnetic labelingMagnetic + cells. For 1.3. table insection details see LS: 3mL Wet filterwith buffer before 130-041-407) to remove Order no. 130-090-532

140-000-563.09 ▲ ▲ ▲ 4. 3. ▲ 7. 6. 5. are for research useonlyandnotfor diagnostic ortherapeutic use. For instructions on column the assembly and separation, the refer to details refer describing to separation cell section the the programs in Magnetic separation with the autoMACS™ the separation with Magnetic Separator or the Columns XS separation with Magnetic magnetic labeling, and of frequency the cells. For labeled magnetically autoMACS Pro Separator should have atemperature of ≥ the respectivethe manual. user autoMACS™the Separator or autoMACS the Pro Separator. Column data XS the sheet. autoMACS™ Pro Separator Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

Program choice dependson isolation the strategy, strength the of Buffers for used operatingthe autoMACSSeparator the or Refer to respective the manual user for instructions on how to use 1 to 6by using column. anew 1. To increase purity of 3. 2. Remove column from separator the and place it on asuitable cells that unlabeled pass throughCollect and wash column with Apply suspension cell onto column. the Pipette appropiate amount of buffer ontothecolumn.

Magnetic separation with the autoMACS™ the separation with Magnetic Pro Separator autoMACS™ the separation with Magnetic Separator Repeat the magneticRepeat the separation procedure in steps as described Immediately flush outcells labeled the magnetically by firmly eluted enriched can over be fraction a second MSor LS Column. collection tube. new buffernew thewhencolumn is empty.reservoir first ontosecond,the equilibrated column instead collection of a tube. pushing plunger the into column. the fraction. cell unlabeled the appropriatethe amount of buffer. bufferthreetimes theoncecolumn is empty.reservoir Only add 3. 2. 1. Note: Collect positive inrow fraction Collect Cof tube the rack. Positive “ selection: For astandard separation following the choose program: Apply tube containing Prepare and prime instrument. the Prepare and prime instrument. the Collect positive from fraction Collect outlet pos2. port Positive “Posseld” selection: For astandard separation following the choose program: Apply tube containing collecting the labeled and fractions. Place labeled cell the collecting unlabeled and fractions. Place labeled cell the collecting unlabeled sample tube inrow Aof tube the rack and collection fraction sample tube at uptake the and collection port fraction the tubes inrows Band C. tubes at neg1 and port pos2. port MS: 3×500µL MS: 1mL

To perform a second column you run, may elute cells directlythe from the CD304 Posseld LS: 5mL LS: 3×3mL

Perform washing steps by adding the samplethe and provide tubes for the samplethe and provide tubes for (BDCA-4/Neuropilin-1) ” Collect total effluent;Collect this is 10 °C. + cells, the cells, the

3. Cells wereCells stained for CD303 (BDCA-2) debris and and CD123. Cell MicroBead Kit,MicroBead two MSColumns, and aMiniMACS™ Separator. dead cells weredead excluded from analysis the on scatter based signals Isolation of CD304(BDCA-4/Neuropilin-1) cells from PBMCs using CD304(BDCA-4/Neuropilin-1) the and PI fluorescence. PI and page 3/4 page (BDCA Example ofExample aseparation using CD304 the ‑4/Neuropilin-1) Kit MicroBead

CD123 CD123 Neuropilin-1) Isolated CD304(BDCA-4/ Before separation CD303 (BDCA-2) CD303 (BDCA-2) dendritic cells cells dendritic + plasmacytoid plasmacytoid + plasmacytoid dendritic Order no. 130-090-532 140-000-563.09 4. 14. 13. 12. 11. 10. 1. Warnings 4. acid, which is extremely toxic. Azide compounds should diluted be with running water are for research useonlyandnotfor diagnostic ortherapeutic use. 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. where explosive conditions may develop. Refer to to Refer contact. 7. 3. 9. 6. 5. Reagents contain azide. sodium Under acidic conditions azide yields sodium hydrazoic Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products 8. before discarding. These precautions are recommended to avoid deposits in plumbing

References

103: 648–655.[4223] Grabbe, S. Krug, A. Krug, Jarrossay, D. Kwakkenbos, M.J. G. Penna, oon, M. Colonna, Kaser,A. Wit, De M. Matsumoto, Penna, G. Dzionek, A. Dzionek, A. Hornung, V. Dzionek, A. and promotes Th1inductionthroughDC2s IL-18by receptor expression. Blood and plasmacytoid dendritic cells. J. Immunol. 169:6673–6676.[2570] Today 21:431–433.[899] 31: 3388–3393.[1278] [4224] [2437] [898] stimulus for plasmacytoid dendritic cells which synergizes with CD40ligand to subsets of dendritic cells inhuman peripheral J. blood. Immunol. 165:6037–6046. plasmacytoid dendritic cells. J. Immunol. 167:1862–1866.[1069] recognition by human myeloid and plasmacytoid dendritic cells. Eur. J. Immunol. CpG oligodeoxynucleotides. J. Immunol. 168:4531–4537.[2385] Immunol. 5:1219–1226.[4292] mediates lectin, antigenII C-type capture and is apotent inhibitor of interferon α/β to specific cellular tofunctions. specific Hum. Immunol. 63:1133–1148.[2423] in cellular subsets of human mononuclear peripheral blood cells and sensitivity to induction. J. Exp. Med. 194:1823–1834.[1264] induce amounts high of IL-12. Eur. J. Immunol. 31:3026–3037.[1215] human dendritic cells. J. Immunol. 171:3154–3162.[4228] heterodimeric receptor expressed on myeloid cells. J. Biol. Leukoc. 71:854–862. www.miltenyibiotec.com et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. (2004) Blood plasmacytoid (2004)Blood 103:1030–1032. dendritic cells. Blood (2001)Toll-like receptor reveals CpG DNA as aunique microbial (2004) Interleukin-18 attracts plasmacytoid dendritic cells (DC2s) et al. (2002)Cutting edge: differential chemokine production by myeloid (2000) Dendritic cells: multi-lineal and multi-functional. Immunol. (2001)Cutting usage edge: selective of chemokine receptors by (2000)BDCA-2, BDCA-3 and BDCA-4: three markers for distinct (2002) Plasmacytoid dendritic cells: from markerssurface specific (2001)BDCA-2, anovel plasmacytoid type dendritic cell–specific et al. (2001) Specialization and complementarity inmicrobial molecule (2002) Quantitative expression of Toll-like receptor 1–10mRNA et al. (2004) Plasmacytoid dendritic cells inimmunity. Nature (2003)Subcellular localization of Toll-like receptor 3in (2002) The human EGF-TM7family member EMR2 is a for all data sheets and protocols. protocols. and sheets data for all

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are registered trademarks or trademarks of . MiniMACS, OctoMACS, All otherAll trademarks Order no. 130-090-532