CD14 MicroBeads - Miltenyi Biotec
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1. Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 4 42. cell labeling A Volumes for magnetic labeling given below are for up to 10 total cells When working with fewer than 10 cells use the same volumes as indicated When working with higher cell numbers scale up all reagent volumes and total volumes accordingly e g for 2x10 total cells use twice the volume of all indicated reagent volumes and total volumes A For optimal performance it is important to obtain a single cell suspension before magnetic separation Pass cells through 30 um nylon mesh Pre Separation Filters 130 041 407 to remove cell clumps which may clog the column Wet filter with buffer before use A Working on ice may require increased incubation times Higher temperatures and or longer incubation times may lead to non specific cell labeling 1 Determine cell number 2 Centrifuge cell suspension at 300xg for 10 minutes Aspirate supernatant completely 3 Resuspend cell pellet in 80 uL of buffer per 10 total cells 4 Add 20 uL of CD14 MicroBeads per 10 total cells 5 Mix well and incubate for 15 minutes in the refrigerator 2 8 C Order no 130 050 201 6 Optional Add staining antibodies e g 10 uL of CD14 FITC 130 080 701 and incubate for 5 minutes in the dark in the refrigerator 2 8 C 7 Wash cells by adding 1 2 mL of buffer per 10 cells and centrifuge at 300xg for 10 minutes Aspirate supernatant completely 8 Resuspend up to 10 cells in 500 uL of buffer
3. pleural peritoneal or synovial fluids or from various tissues such as spleen and lymph node The CD14 antigen belongs to the LPS receptor complex Binding of antibody to CD14 does not trigger signal transduction since CD14 lacks a cytoplasmatic domain CD14 is strongly expressed on most monocytes and macrophages and weakly on neutrophils and some myeloid dendritic cells CD14 MicroBeads human Order no 130 050 201 1 3 Applications Isolation of CD14 monocytes for in vitro generation of dendritic cells or macrophages Isolation of CD14 monocytes for studies on cytotoxicity and migration 1 4 Reagent and instrument requirements e Buffer Prepare a solution containing phosphate buffered saline PBS pH 7 2 0 5 bovine serum albumin BSA and 2mM EDTA by diluting MACS BSA Stock Solution 130 091 376 1 20 with autoMACS Rinsing Solution 130 091 222 Keep buffer cold 2 8 C Degas buffer before use as air bubbles could block the column A Note EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula A ACD A or citrate phosphate dextrose CPD BSA can be replaced by other proteins such as human serum albumin human serum or fetal A toe 2 2 bovine serum Buffers or media containing Ca or Mg are not recommended for use MACS Columns and MACS Separators CD14 cells can be enriched by using MS LS or XS Columns or depleted with the use of LD CS o
4. 90 Z90 000 0 L Miltenyi Biotec Contents 1 Description 1 1 Principle of the MACS Separation 1 2 Background information 1 3 1 4 Applications Reagent and instrument requirements 2 Protocol 2 1 Sample preparation 2 2 Magnetic labeling 2 3 Magnetic separation 3 Example of a separation using the CD14 MicroBeads 4 References 1 Description Components 2 mL CD14 MicroBeads human MicroBeads conjugated to monoclonal anti human CD14 antibodies isotype mouse IgG2a Capacity For 10 total cells up to 100 separations Product format CD14 MicroBeads are supplied in buffer containing stabilizer and 0 05 sodium azide Storage Store protected from light at 2 8 C Do not freeze The expiration date is indicated on the vial label 1 1 Principle of the MACS Separation First the CD14 cells are magnetically labeled with CD14 MicroBeads Then the cell suspension is loaded onto a MACS Column which is placed in the magnetic field of a MACS Separator The magnetically labeled CD14 cells are retained within the column The unlabeled cells run through this cell fraction is thus depleted of CD14 cells After removing the column from the magnetic field the magnetically retained CD14 cells can be eluted as the positively selected cell fraction 1 2 Background information CD14 MicroBeads are used for the positive selection or depletion of human monocytes and macrophages from cord blood or PBMCs as well as
5. A Note For higher cell numbers scale up buffer volume accordingly A Note For depletion with LD Columns resuspend up to 1 25x10 cells in 500 uL of buffer 9 Proceed to magnetic separation 2 3 Ae 2 3 Magnetic separation A Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD14 cells For details see table in section 1 4 Magnetic separation with MS or LS Columns 1 Place column in the magnetic field of a suitable MACS Separator For details see the respective MACS Column data sheet 2 Prepare column by rinsing with the appropriate amount of buffer MS 500 pL LS 3 mL 3 Apply cell suspension onto the column 4 Collect unlabeled cells that pass through and wash column with the appropriate amount of buffer Collect total effluent this is the unlabeled cell fraction Perform washing steps by adding buffer three times Only add new buffer when the column reservoir is empty MS 3x500 uL LS 3x3 mL 5 Remove column from the separator and place it on a suitable collection tube 6 Pipette the appropriate amount of buffer onto the column Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column MS 1 mL LS 5 mL 7 Optional To increase the purity of CD14 cells the eluted fraction can be enriched over a second MS or LS Column Repeat the magnetic separation procedure as described in steps 1 to 6 by using a n
6. d C Order no 130 050 201 3 For a standard separation choose one of the following programs Positive selection Possel Collect positive fraction in row C of the tube rack Depletion Depletes Collect negative fraction in row B of the tube rack 3 Example of a separation using the CD14 MicroBeads CD14 monocytes were isolated from human PBMCs using CD14 MicroBeads an MS Column and a MiniMACS Separator Cells are fluorescently stained with CD14 FITC 130 080 701 Cell debris and dead cells are excluded from the analysis based on scatter signals and PI fluorescence PBMCs before separation Relative cell number CD14 FITC CD14 cells T 2 E 5 lt ir v v 2 5 o T 4 CD14 FITC CD14 cells T 2 E 5 lt oO v v 2 S T cc CD14 FITC Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 3 4 90 Z90 000 0 L 4 References 1 Pickl W F et al 1996 Molecular and Functional Characteristics of Dendritic Cells Generated from Highly Purified CD14 Peripheral Blood Monocytes J Immunol 157 3850 3859 274 2 Hanley P J et al 2004 Extracellular ATP induces oscillations of intracellular Ca2 and membrane potential and promotes transcription of IL 6 in macrophages Proc Natl Acad Sci U S A 101 9479 9484 4206 3 Verreck F A et al 2004 Human IL 23 p
7. ew column Magnetic separation with XS Columns For instructions on the column assembly and the separation refer to the XS Column data sheet Depletion with LD Columns 1 Place LD Column in the magnetic field of a suitable MACS Separator For details see LD Column data sheet 2 Prepare column by rinsing with 2 mL of buffer 3 Apply cell suspension onto the column Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 2 4 90 Z90 000 0 L 4 Collect unlabeled cells that pass through and wash column with 2xlmL of buffer Collect total effluent this is the unlabeled cell fraction Perform washing steps by adding buffer two times Only add new buffer when the column reservoir is empty Depletion with CS Columns 1 Assemble CS Column and place it in the magnetic field of a suitable MACS Separator For details see CS Column data sheet 2 Prepare column by filling and rinsing with 60 mL of buffer Attach a 22G flow resistor to the 3 way stopcock of the assembled column For details see CS Column data sheet 3 Apply cell suspension onto the column 4 Collect unlabeled cells that pass through and wash column with 30 mL buffer from the top Collect total effluent this is the unlabeled cell fraction Depletion with D Columns For instructions on column assembly and separation refer to the D Column data sheet Mag
8. netic separation with the autoMACS Separator or the autoMACS Pro Separator A Refer to the respective user manual for instructions on how to use the autoMACS Separator or the auttoMACS Pro Separator A Buffers used for operating the autoMACS Separator or the autoM ACS Pro Separator should have a temperature of gt 10 C A Program choice depends on the isolation strategy the strength of magnetic labeling and the frequency of magnetically labeled cells For details refer to the section describing the cell separation programs in the respective user manual Program recommendations below refer to separation of human PBMCs Magnetic separation with the autoMACS Separator 1 Prepare and prime the instrument 2 Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions Place sample tube at the uptake port and the fraction collection tubes at port neg and port posl 3 For a standard separation choose one of the following programs Positive selection Possel Collect positive fraction from outlet port posl Depletion Depletes Collect negative fraction from outlet port negl Magnetic separation with the autoMACS Pro Separator 1 Prepare and prime the instrument 2 Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions Place sample tube in row A of the tube rack and fraction collection tubes in rows B an
9. ptional Propidium iodide PI or 7 AAD for flow cytometric exclusion of dead cells Optional Dead Cell Removal Kit 130 090 101 for the depletion of dead cells Optional Pre Separation Filters 130 041 407 to remove cell clumps 2 Protocol 2 1 Sample preparation When working with anticoagulated peripheral blood or buffy coat peripheral blood mononuclear cells PBMCs should be isolated by density gradient centrifugation for example using Ficoll Paque For details see the General Protocols section of the respective separator user manual The General Protocols are also available at www miltenyibiotec com protocols A Note To remove platelets after density gradient separation resuspend cell pellet in buffer and centrifuge at 200xg for 10 15 minutes at 20 C Carefully aspirate supernatant Repeat washing step When working with tissues or lysed blood prepare a single cell suspension using standard methods For details see the General Protocols section of the respective separator user manual The General Protocols are also available at www miltenyibiotec com protocols A Dead cells may bind non specifically to MACS MicroBeads To remove dead cells we recommend using density gradient centrifugation or the Dead Cell Removal Kit 130 090 101 a 2 2 Magnetic labeling A Work fast keep cells cold and use pre cooled solutions This will prevent capping of antibodies on the cell surface and non specific
10. r D Columns Cells which strongly express the CD14 antigen can also be depleted using MS LS or XS Columns Positive selection or depletion can also be performed by using the autoMACS or the autoMACS Pro Separator Column Max number Max number Separator of labeled cells of total cells Positive selection MS 107 2x10 MiniMACS OctoMACS VarioMACS SuperMACS LS 10 2x10 MidiMACS QuadroMACS VarioMACS SuperMACS XS 10 2x10 SuperMACS Depletion LD 10 5x10 MidiMACS QuadroMACS VarioMACS SuperMACS CS 2x10 VarioMACS SuperMACS D 10 SuperMACS Positive selection or depletion autoMACS 2x10 4x10 autoMACS autoMACS Pro A Note Column adapters are required to insert certain columns into the VarioMACS or SuperMACS Separators For details see the respective MACS Separator data sheet Optional Fluorochrome conjugated CD14 antibody for flow cytometric analysis e g CD14 FITC 130 080 701 CD14 PE 130 091 242 or CD14 APC 130 091 243 For more information about other fluorochrome conjugates see www miltenyibiotec com Miltenyi Biotec GmbH Friedrich Ebert StraBe 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 macs miltenyibiotec de www miltenyibiotec com Miltenyi Biotec Inc 2303 Lindbergh Street Auburn CA 95602 USA Phone 800 FOR MACS 1 530 888 8871 Fax 1 530 888 8925 macs miltenyibiotec com page 1 4 90 Z90 000 0 L O
11. receptor 3 in human dendritic cells J Immunol 171 3154 3162 4228 Erratum color print of figure 5A in J Immunol 171 4934 Order no 130 050 201 All protocols and data sheets are available at www miltenyibiotec com Warnings Reagents contain sodium azide Under acidic conditions sodium azide yields hydrazoic acid which is extremely toxic Azide compounds should be diluted with running water before discarding These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop Warranty The products sold hereunder are warranted only to be free from defects in workmanship and material at the time of delivery to the customer Miltenyi Biotec GmbH makes no warranty or representation either expressed or implied with respect to the fitness of a product for a particular purpose There are no warranties expressed or implied which extend beyond the technical specifications of the products Miltenyi Biotec GmbH s liability is limited to either replacement of the products or refund of the purchase price Miltenyi Biotec GmbH is not liable for any property damage personal injury or economic loss caused by the product MACS isa registered trademark and autoM ACS MidiMACS MiniMACS OctoMACS QuadroMACS SuperMACS and VarioMACS are trademarks of Miltenyi Biotec GmbH Ficoll Paque is a trademark of GE Healthcare companies 2007 Miltenyi Biotec GmbH Unless otherwise specifically indicated
12. roducing type 1 macrophages promote but IL 10 producing type 2 macrophages subvert immunity to myco bacteria Proc Natl Acad Sci U S A 101 4560 4565 4198 4 Ryan E J et al 2002 Dendritic cell associated lectin 1 a novel dendritic cell associated C type lectin like molecule enhances T cell secretion of IL 4 J Immunol 169 5638 5648 2436 5 Vitale S et al 2004 Soluble fractalkine prevents monocyte chemoattractant protein 1 induced monocyte migration via inhibition of stress activated protein kinase 2 p38 and matrix metalloproteinase activities J Immunol 172 585 592 3657 6 de Baey A and Lanzavecchia A 2000 The Role of Aquaporins in Dendritic Cell Macropinocytosis J Exp Med 191 743 747 841 7 Salio M et al 2000 Dendritic cell maturation is induced by mycoplasma infection but not by necrotic cells Eur J Immunol 30 705 708 973 8 Ebner S et al 2002 A novel role for IL 3 human monocytes cultured in the presence of IL 3 and IL 4 differentiate into dendritic cells that produce less IL 12 and shift Th cell responses toward a Th2 cytokine pattern J Immunol 168 6199 6207 2200 9 Jefford M et al 2003 Functional comparison of DCs generated in vivo with Fit3 ligand or in vitro from blood monocytes differential regulation of function by specific classes of physiologic stimuli Blood 102 1753 1763 2874 10 Matsumoto M et al 2003 Subcellular localization of Toll like
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