140-000-067.07 1. 1.2 1.1 1. Description 4. The unlabeled cells run through; this cell fraction is thus depleted depleted thus is fraction cell this through; run cells unlabeled The CD14 labeled magnetically The

Contents 2. Capacity Components 3. CD14 MicroBeads are used for the positive selection or depletion of or depletion selection positive for the used CD14 are MicroBeads Separator. of aMACS field magnetic the in placed is which Column MicroBeads. Then, the cell suspension is loaded onto a MACS® onto aMACS® loaded is suspension cell the Then, MicroBeads. weakly on and some myeloid dendritic cells. cells. myeloid some dendritic and on neutrophils weakly tissues, various or from fluids or synovial peritoneal, pleural, as well First, the CD14 the First, of CD14 of Product format LPS complex. Binding of antibody to CD14 does not trigger CD14 to not trigger of antibody does Binding complex. receptor LPS macsde since CD14 lacks a cytoplasmatic domain. CD14 domain. CD14 acytoplasmatic since lacks transduction signal CD14 the to The belongs node. lymph antigen and spleen as such fraction. cell selected www.miltenyibiotec.com www.miltenyibiotec.com Storage human and from cord blood or PBMCs, as as or PBMCs, blood cord from macrophages and monocytes human the magnetically retained CD14 retained magnetically the is strongly expressed on most monocytes and macrophages and and macrophages and monocytes on most expressed strongly is 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 CD14 the using MicroBeads 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 +49 2204 8306-0, 2204 +49 @

+ miltenyi.com cells. After removing the column from the magnetic field, field, magnetic the from column the removing After cells.

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

+ cells are magnetically labeled with CD14 with labeled magnetically are cells For 10⁹ total cells, up to 100 separations. up100 to cells, For 10⁹ total Store protected from at 2−8 light from Store protected human: CD142 mL MicroBeads, CD14 MicroBeads are supplied in buffer buffer in supplied CD14 are MicroBeads vial label. vial MicroBeads conjugated to monoclonal anti- 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 the indicated is date expiration The freeze. human CD14 antibodies (isotype: mouse mouse (isotype: CD14human antibodies 68, 5142968, Fax Fax +49 2204 85197 2204 +49 Bergisch Gladbach, Germany Bergisch Gladbach, + cells are retained within the column. column. the within retained are cells + cells can be eluted as the positively positively the as eluted be can cells °C. Do not Do °C.

1.4 Applications 1.3 human CD14 MicroBeads

● ● ● ● ● page 1/4

autoMACS depletion or selection Positive D CS LD Depletion XS LS MS selection Positive Column 10⁹ 10⁸ 10⁹ 10⁸ VarioMACS™ or SuperMACS™ Separators. For details see the respective MACS MACS respective the see details For Separators. SuperMACS™ or VarioMACS™ MACS Columns and MACS Separators: CD14 Separators: MACS and Columns MACS ▲ phosphate-buffered containing asolution Prepare Buffer: (Optional) Fluorochrome-conjugated CD14 for antibody Fluorochrome-conjugated (Optional) ▲ Isolation of CD14 of Isolation CD14 of Isolation Reagent and instrument requirements instrument and Reagent 222). Keep buffer cold (2−8 cold 222). buffer Keep 2 10⁷ dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be be (CPD). can BSA dextrose phosphate (ACD-A) citrate or formula-A dextrose CD14-PE (# performed be also can or depletion selection Positive Columns. www.miltenyibiotec.com. dendritic cells¹ or macrophages² or cells¹ dendritic enriched by using MS, LS, or XS Columns or depleted with with or depleted Columns or XS LS, MS, by using enriched replaced by other such as human serum albumin, human serum, or fetal fetal or serum, human albumin, serum human as such proteins other by replaced migration⁵. more information about other fluorochrome-conjugates see see fluorochrome-conjugates other about more information CD14-FITC e.g., (# analysis, cytometric flow saline (PBS), pH saline use. 091-376) 1:20 with autoMACS™ Rinsing Solution (# Solution 091-376) Rinsing autoMACS™ with 1:20 the CD14 antigen can also be depleted using MS, LS, or XS or XS LS, MS, using depleted be also CD14 the can antigen express strongly which Cells or D Columns. of LD, CS, use the bubbles could block the column. the block could bubbles by using the autoMACS or the autoMACS Pro Separator. Separator. Pro autoMACS or the autoMACS the by using Separator data sheet. data Separator bovine serum. Buffers or media containing Ca containing media or Buffers serum. bovine

mM EDTA by diluting MACS BSA Stock Solution (# Solution Stock BSA MACS EDTA by diluting mM Note: Note:

2×10⁸ cells labeled of EDTA can be replaced by other supplements such as anticoagulant citrate citrate anticoagulant as such supplements other by replaced be can EDTA number Max. Column adapters are required to insert certain columns into the the into columns certain insert to required are adapters Column 2×10⁸ 130-091-242), or CD14-APC (#

+ 7.2, 0.5% bovine serum albumin (BSA), and albumin serum 7.2, bovine 0.5%

monocytes for studies on cytotoxicity⁴ and and on cytotoxicity⁴ for studies monocytes +

monocytes for monocytes Max. number Max. of total cells total of 2 5 2 4 2 ×10¹⁰ ×10⁸ ×10⁸ ×10⁹ ×10⁹ °C). Degas buffer before use, as air air as use, before buffer °C). Degas

, ³. Order no. 130-050-201

2+ or Mg or SuperMACS SuperMACS VarioMACS, SuperMACS VarioMACS, QuadroMACS, MidiMACS, SuperMACS SuperMACS VarioMACS, QuadroMACS, MidiMACS, SuperMACS VarioMACS, MiniMACS, OctoMACS, Separator autoMACS, autoMACS Pro autoMACS autoMACS, in vitro in 2+ are not recommended for for recommended not are 130-091-243). For generation of of generation + cells can be be can cells 130-080-701), 130-091- 130-

140-000-067.07 ▲ ▲ ▲ ▲ ▲ 1. same the use 10⁷ cells, than fewer with working When cells. 10⁷ total 4. When working with tissues or lysed blood, prepare a single-cell asingle-cell prepare blood, or lysed tissues with working When coat, or buffy blood peripheral anticoagulated with working When 2. 3.

2.1 2. ● ● ● 5. volumes and total volumes). total and volumes numbers, cell higher with working When indicated. as volumes General Protocols are also available at www.miltenyibiotec.com/ available also are Protocols General are for research useonlyandnotfor diagnostic ortherapeutic use. www.miltenyibiotec.com/protocols. Protocols section of the respective separator user manual. The The manual. user separator respective of the section Protocols respective of the section Protocols General the see For details or the Dead Cell Removal Kit (# Kit 130-090-101). Removal Cell Dead or the density gradient centrifugation, for example, using Ficoll-Paque™. Ficoll-Paque™. using for example, centrifugation, gradient density clumps which may clog the column. Wet filter with buffer before before buffer with Wet filter column. the clog may which clumps labeling. cell remove dead cells, we recommend using density gradient centrifugation centrifugation gradient density using we recommend cells, remove dead use. # Filters, (Pre-Separation 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 specific cell labeling. cell specific 30 through cells Pass separation. magnetic before suspension (e.g. accordingly volumes total and volumes reagent up all scale General the see For details methods. standard using suspension at available also are Protocols General The manual. user separator temperatures and/or longer incubation times may lead to non- to lead may times incubation longer and/or temperatures non-specific and surface cell on the of antibodies capping prevent protocols. by (PBMCs) isolated be should cells mononuclear blood peripheral Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

Work fast, keep cells cold, and use pre-cooled solutions. This will will This solutions. pre-cooled use and cold, cells keep Work fast, Working on ice may require increased incubation times. Higher Higher times. incubation increased require may on ice Working For optimal performance it is important to obtain asingle obtain to important it is performance For optimal Dead cells may bind non-specifically to MACS MicroBeads. To MicroBeads. MACS to non-specifically bind may cells Dead Volumes for magnetic labeling given below are for up to are below given labeling Volumes for magnetic ▲ (# Filters Pre-Separation (Optional) (# Kit Removal Cell Dead (Optional) cytometric (PI) or 7-AAD for flow iodide Propidium (Optional) Protocol Mix well and incubate for 15 incubate and well Mix cells. 10⁷ of CD14 per total µL Add 20 MicroBeads for 10 at 300×g suspension cell Centrifuge µL of buffer per 10⁷ total cells. cells. 10⁷ per total of buffer µL 80 in pellet cell Resuspend Determine cell number. number. cell Determine Sample preparation Sample (2−8 aspirate supernatant. Repeat step. washing depletion of cells. dead cell clumps. cell cells. of dead exclusion pellet in buffer and centrifuge at 200×g for 10−15 for 200×g at centrifuge and buffer in pellet supernatant completely. supernatant

Note: °C). °C).

To remove platelets after density gradient separation, resuspend cell cell resuspend separation, gradient density after To platelets remove 2.2 Magnetic labeling Magnetic minutes in the refrigerator refrigerator the in minutes 130-041-407) to remove cell remove to cell 130-041-407) 130-041-407) to remove to 130-041-407) 20 at minutes 130-090-101) for the minutes. Aspirate Aspirate minutes. °C. Carefully Carefully °C. ‑

cell cell µm µm

▲ 1. 1. 4. 3. 3.

2. 2. 7. 7. 9. 6. 6. 5. For details see table in section 1.4. section in table see For details For instructions on the column assembly and the separation refer to to refer separation the and assembly column on the For instructions Magnetic separation with MS or LS Columns or LS MS with separation Magnetic Magnetic separation with XS Columns Columns XS with separation Magnetic Depletion with LD Columns Columns LD with Depletion 8. according to the number of total cells and the number of CD14 number the and cells of total number the to according the XS Column data sheet. sheet. data Column XS the page 2/4 page Choose an appropriate MACS Column and MACS Separator Separator MACS and Column MACS appropriate an Choose Proceed to magnetic separation (2.3). separation magnetic to Proceed Resuspend up to 10⁸ cells in 500 µL of buffer. µL 500 in up10⁸ to cells Resuspend ▲ ▲ Apply cell suspension onto the column. column. onto the suspension Apply cell of amount appropriate the with by rinsing column Prepare Apply cell suspension onto the column. onto the suspension Apply cell of buffer. 2 mL 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 column wash and through pass that cells unlabeled Collect (Optional) Add staining antibodies, e.g., 10 e.g., antibodies, Add staining (Optional) Pipette the appropriate amount of buffer onto the column. column. onto the of buffer amount appropriate the Pipette MACS of asuitable field magnetic the in column Place 1−2 by adding Wash cells Place LD Column in the magnetic field of a suitable MACS MACS of a suitable field magnetic the in Column LD Place of CD14 purity the To(Optional) increase (# with the appropriate amount of buffer. Collect total effluent; effluent; total Collect of buffer. amount appropriate the with Repeat the magnetic separation procedure as described in in described as procedure separation magnetic the Repeat by firmly cells labeled magnetically out the flush Immediately completely. for 10 at 300×g centrifuge collection tube. empty. is reservoir column refrigerator (2−8 °C). refrigerator fraction can be enriched over a second MS or LS Column. Column. or LS MS over asecond enriched be can fraction sheet. data Column MACS respective the see For details Separator. Separator. For details see LD Column data sheet. data Column LD see For details Separator. column. anew 6by 1to using steps adding buffer three times. Only add new buffer when the the when buffer new add Only times. three buffer adding µL of buffer. of µL 500 pushing the plunger into the column. the into plunger the pushing by steps washing Perform fraction. cell unlabeled the is this buffer:

Note: Note: 130-080-701), and incubate for 5 130-080-701), incubate and 2.3 µL 500 MS: 1mL MS: µL 3×500 MS:

For higher cell numbers, scale up buffer volume accordingly. accordingly. volume buffer up scale numbers, cell higher For 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 Magnetic separation Magnetic LS: 3mL LS: LS: 5mL LS: 3×3 mL LS: mL of buffer per 10⁷ cells and and 10⁷ per cells of buffer mL minutes. Aspirate supernatant supernatant Aspirate minutes. minutes in the dark in the the in dark the in minutes + µL of CD14-FITCµL cells, the eluted eluted the cells, Order no. 130-050-201 no. Order + cells.

140-000-067.07 ▲ ▲ ▲ 1. 4. 4. 3.

2. D Column data sheet. sheet. data D Column are for research useonlyandnotfor diagnostic ortherapeutic use. For instructions on column assembly and separation refer to the the to refer separation and assembly on column For instructions 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 Magnetic separation with the autoMACS™ Separator or the or the Separator autoMACS™ the with separation Magnetic Depletion with CS Columns Columns CS with Depletion Depletion with D Columns DColumns with Depletion use the autoMACS™ Separator or the autoMACS Pro Separator. Pro autoMACS or the Separator autoMACS™ the use autoMACS Pro Separator should have a temperature of ≥ have atemperature should Separator Pro autoMACS programs in the respective user manual. Program recommendations recommendations Program manual. user respective the in programs autoMACS™ Pro Separator Pro autoMACS™ Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products below refer to separation of human PBMCs. of human separation to refer below

Program choice depends on the isolation strategy, the strength strength the strategy, isolation on the depends choice Program or the Separator autoMACS the for operating used Buffers on how to for instructions manual user respective the to Refer Attach a 22G flow resistor to the 3-way stopcock of the of the stopcock 3-way the to resistor flow a22G Attach 1. 3. 2. 2. 1. Collect unlabeled cells that pass through and wash column column wash and through pass that cells unlabeled Collect wash and through pass that cells unlabeled Collect Apply cell suspension onto the column. onto the suspension Apply cell 60 with rinsing and by filling column Prepare Assemble CS Column and place it in the magnetic field of a field magnetic the it in place and Column CS Assemble Magnetic separation with the autoMACS™ Separator autoMACS™ the with separation Magnetic Magnetic separation with the autoMACS™ Pro Separator Pro autoMACS™ the with separation Magnetic 30 with column with 2×1 with column sheet. data Column CS see For details Separator. MACS suitable when buffer new add Only times. two buffer by adding steps assembled column. For details see CS Column data sheet. data Column CS see For details column. assembled the column reservoir is empty. is reservoir column the washing Perform fraction. cell unlabeled the is this the unlabeled cell fraction. cell unlabeled the 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 Apply tube containing Apply tube instrument. the prime and Prepare neg1. port outlet from fraction negative Collect “Depletes” Depletion: pos1. port outlet from fraction positive Collect “Possel” selection: Positive Place sample tube in row A of the tube rack and fraction fraction and rack tube row Aof the in tube sample Place collection tubes in rows B and C. Band rows in tubes collection Place fractions. cell unlabeled and labeled the collecting for collecting the labeled and unlabeled cell fractions. fractions. cell unlabeled and labeled the for collecting sample tube at the uptake port and the fraction collection collection fraction the and port uptake at the tube sample programs: pos1. port neg1 and at port tubes mL buffer from the top. Collect total effluent; this is is this effluent; total top. Collect the from buffer mL mL of buffer. Collect total effluent; effluent; total Collect of buffer. mL

the sample and provide tubes for tubes provide and sample the

the sample and provide tubes tubes provide and sample the mL of buffer. of buffer. mL °C. 10 °C.

3.

CD14 CD14 MicroBeads, an MS Column, and a MiniMACS™ Separator. Cells Cells Separator. aMiniMACS™ and Column, MS an MicroBeads, debris and dead cells are excluded from the analysis based on scatter on scatter based analysis the from excluded are cells dead and debris signals and PI fluorescence. and signals are fluorescently stained with CD14-FITC (# with stained fluorescently are page 3/4 page 3. Example of a separation using the the using of aseparation Example CD14 MicroBeads + monocytes were isolated from human PBMCs using CD14 PBMCs using human from were isolated monocytes Collect negative fraction in row B of the tube rack. tube row Bof the in fraction negative Collect Depletion: “ Depletion: rack. tube row Cof the in fraction positive Collect “ selection: Positive following one of the choose separation For astandard programs:

Relative cell number Relative cell number Relative cell number Depletes PBMCs before separation Possel ” CD14 CD14-FIT CD14-FIT CD14-FIT ” + – cells cells C C C

130-080-701). Cell 130-080-701). Cell Order no. 130-050-201 no. Order 140-000-067.07 References4. 10. 1. Warnings 4. deposits in plumbing where explosive conditions may develop. may conditions explosive where plumbing in deposits 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. Refer to to Refer contact. 7. 3. 9. 6. running water before discarding. These precautions are recommended to avoid avoid to recommended are precautions These discarding. before water running 5. Reagents contain sodium azide. Under acidic conditions sodium azide yields yields azide sodium conditions acidic Under azide. sodium contain Reagents Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products 8. hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with with diluted be should compounds Azide toxic. extremely is which acid, hydrazoic 12 and shift Th cell responses toward a Th2 pattern. J. Immunol. 168: 168: Immunol. J. pattern. cytokine aTh2 toward responses cell Th shift and 12 Ryan, E. J. J. E. Ryan, Hanley, P. J. Hanley, Ebner, S. S. Ebner, Jefford, M. M. Jefford, F. A. Verreck, Matsumoto, M. M. Matsumoto, W. F. Pickl, de Baey, A. and Lanzavecchia, A. (2000) The Role of Aquaporins in Dendritic Dendritic in Aquaporins of Role The (2000) A. Lanzavecchia, and A. Baey, de Salio, M. M. Salio, Vitale, S. S. Vitale, cell-associated, C-type lectin-like molecule enhances secretion of IL-4. J. J. IL-4. of secretion Tcell enhances molecule lectin-like C-type cell-associated, of figure 5A) in: J. Immunol. 171: 4934. 171: 4934. Immunol. J. 5A) in: figure of macrophages. Proc. Natl. Acad. Sci. U S A. 101: 9479–9484. [4206] 101: USA. 9479–9484. Sci. Acad. Natl. Proc. macrophages. [3657] presence of IL-3 and IL-4 differentiate into dendritic cells that produce less IL- less produce that cells dendritic into differentiate IL-4 and IL-3 of presence stress-activated of inhibition via migration protein-1-induced 6199–6207. [2200] 6199–6207. Cell Macropinocytosis. J. Exp. Med. 191: 743–747. Med. Exp. J. [841] Macropinocytosis. Cell in IL-6 of transcription promotes and potential membrane and Ca2+ CD14 Purified Highly from Generated Cells Flt3 ligand or in vitro from blood monocytes: differential regulation of function function of regulation differential monocytes: blood from vitro in or ligand Flt3 [4198] 101: USA. 4560–4565. Sci. Acad. Natl. Proc. Immunol. 169: 5638–5648. [2436] 169: 5638–5648. Immunol. [274] 157: 3850–3859. Immunol. infection but not by necrotic cells. Eur. J. Immunol. 30: 705–708. [973] 705–708. 30: Immunol. J. Eur. cells. necrotic by not but infection kinase 2/p38 and matrix metalloproteinase activities. J. Immunol. 172: 585–592. 172: 585–592. Immunol. J. activities. metalloproteinase matrix and 2/p38 kinase by specific classes of physiologic stimuli. Blood 102: 1753–1763. 102: Blood [2874] stimuli. physiologic of classes specific by (myco)bacteria. to immunity subvert 2macrophages type IL-10-producing but human dendritic cells. J. Immunol. 171: 3154–3162. [4228] Erratum (color print (color print 171: Erratum 3154–3162. [4228] Immunol. J. cells. dendritic human www.miltenyibiotec.com et al. et et al. al. et et al. et et al. al. et et al. et et al. al. et et al. al. et et al. al. et (2002) A novel role for IL-3: human monocytes cultured in the the in cultured monocytes human IL-3: for role Anovel (2002) (2000) maturation is induced by mycoplasma mycoplasma by induced is maturation cell Dendritic (2000) (2004) Soluble fractalkine prevents monocyte chemoattractant chemoattractant monocyte prevents fractalkine Soluble (2004) et al. al. et (1996) Molecular and Functional Characteristics of Dendritic Dendritic of Characteristics Functional and (1996) Molecular (2003) Functional comparison of DCs generated in vivo with with vivo in generated DCs of comparison Functional (2003) (2004) Extracellular ATP induces oscillations of intracellular intracellular of oscillations ATPinduces Extracellular (2004) (2002) Dendritic cell-associated lectin-1: a novel dendritic dendritic anovel lectin-1: cell-associated Dendritic (2002) (2004) Human IL-23-producing type 1 macrophages promote promote 1macrophages type IL-23-producing Human (2004) (2003) Subcellular localization of Toll-like receptor 3 in 3 in receptor Toll-like of localization Subcellular (2003) for all data sheets and protocols. protocols. and sheets data for all + Peripheral Blood Monocytes. J. J. Monocytes. Blood Peripheral

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