CD49b (DX5) MicroBeads
Contents
1. 0 0 L 000 0rL Magnetic cell sorting Index 1 Description 1 1 Principle of MACS separation 1 2 Background and product applications 1 3 Reagent and instrument requirements 2 Protocol 2 1 Sample preparation 2 2 Magnetic labeling 2 3 Magnetic separation Example of a separation using CD49b DX5 MicroBeads 4 References 1 Description Components 2 mL CD49b DX5 MicroBeads mouse MicroBeads conjugated to monoclonal anti mouse NK cell DX5 isotype rat IgM clone DX5 antibody Size For 2x10 total cells up to 200 separations Product format CD49b DX5 MicroBeads are supplied as a suspension containing stabilizer and 0 05 sodium azide Storage Store protected from light at 4 8 C Do not freeze The expiration date is indicated on the vial label 1 1 Principle of MACS separation First the CD49b cells are magnetically labeled with CD49b DX5 MicroBeads Then the cell suspension is loaded onto a MACS Column whichis placed in the magnetic field ofa MACS Separator The magnetically labeled CD49b cells are retained on the column The unlabeled cells run through and this cell fraction is depleted of CD49b cells After removal of the column from the magnetic field the magnetically retained NK cells can be eluted as the positively selected cell fraction 1 2 Background and product applications Mouse CD49b DX5 MicroBeads were developed for the isolation of mouse NK cells from single cell suspensio2. en cell suspension using CD49b DX5 MicroBeads a MiniMACS Separator and an MS Column The cells are fluorescently stained with CD49b DX5 FITC 130 091 814 Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence CD49b DX5 cells Relative cell number daar Psi al Spleen cells before separation CD49b DX5 FITC 4 References 1 Tomasello E Desmoulins P O Chemin K Guia S Cremer H Ortaldo J Love P Kaiserlian D Vivier E 2000 Combined Natural Killer Cell and Dendritic Cell Functional Deficiency in KARAP DAP12 Loss of Function Mutant Mice Immunity 13 335 364 945 2 Shi FD Takeda K Akira S Sarvetnick N Ljunggren HG 2000 IL 18 Directs Autoreactive T Cells and Promotes Autodestruction in the Central Nervous System Via Induction of IFN y by NK Cells J Immunol 165 3099 3104 1021 3 Mihlen KA Sch mann J Wittke F Stenger S van Rooijen N van Kaer L Tiegs G 2004 NK cells but not NKT cells are involved in Pseudomonas aeruginosa exotoxin a induced hepatotoxicity in mice J Immunol 172 3034 3041 4274 4 Hsieh GC Loukas A Wahl AM Bhatia M Wang Y et al 2004 A secreted protein from the human hookworm necator americanus binds selectively to NK cells and induces IFN y production J Immunol 173 2699 2704 4275 Miltenyi Biotec This MACS product is for in vitro research use only and not for diagnostic or therapeut
3. fuge cell suspension at 300xg for 10 minutes Pipette off supernatant completely 3 Resuspend cell pellet in 90 uL of buffer per 107 total cells 4 Add 10 uL of CD49b DX5 MicroBeads per 10 total cells 5 Mix well and incubate for 15 minutes at 4 8 C A Note Working on ice may require increased incubation times Higher temperatures and or longer incubation times lead to non specific cell labeling 6 Optional Add a fluorochrome conjugated CD49b antibody according to manufacturer s recommendation and incubate for 5 minutes at 4 8 C 7 Wash cells by adding 1 2 mL of buffer per 10 cells and centrifuge at 300xg for 10 minutes Pipette off supernatant completely 8 Resuspend up to 108 cells in 500 uL of buffer A Note For higher cell numbers scale up buffer volume accordingly A Note For depletion with LD Columns resuspend cell pellet in 500 uL of buffer for up to 1 25x108 cells 9 Proceed to magnetic separation 2 3 Miltenyi Biotec This MACS product is for in vitro research use only and not for diagnostic or therapeutic procedures Order No 130 052 501 nim 2 3 Magnetic separation A Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD49b cells see table in section 1 3 Magnetic separation with MS or LS Columns 1 Place column in the magnetic field of a suitable MACS Separator see Column data sheets 2 Prepare column by rin
4. ic procedures Order No 130 052 501 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 is a registered trademark of Miltenyi Biotec GmbH page 3 3
5. n by filling and rinsing with 60 mL of buffer Attach a 22G flow resistor to the 3 way stopcock of the assembled column see CS Column data sheet 3 Apply cell suspension onto the column 4 Collect unlabeled cells which 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 page 2 3 0 0 L 000 0rL Magnetic separation with the autoMACS Separator A Refer to the autoMACS User Manual for instructions on how to use the autoMACS Separator 1 Prepare and prime autoMACS Separator 2 Place tube containing the magnetically labeled cells in the auttoMACS Separator For a standard separation choose following separation programs Positive selection Possel Depletion Depletes A Note Program choice depends on the isolation strategy the strength of magnetic labeling and the frequency of magnetically labeled cells For details see autoMACS User Manual autoMACS Cell Separation Programs 3 When using the program Possel collect positive fraction outlet port pos1 This is the purified CD49b cell fraction When using the program Depletes collect unlabeled fraction outlet port neg1 This is the CD49b cell fraction 3 Example of a separation using CD49b DX5 MicroBeads NK cells were isolated from a mouse sple
6. ns of lymphoid and non lymphoid tissue as well as from peripheral blood or body fluids The MicroBeads were formerly called Anti NK Cell DX5 MicroBeads CD49b is expressed on NK cells and a small population of T cells CD4 CD3 TCRaB These cells can be depleted prior to NK cell enrichment by using CD90 Thy1 2 MicroBeads or CD5 MicroBeads CD49b is less mouse strain specific than other NK cell markers e g NK1 1 and is expressed by the most common inbred mouse strains Examples of applications Isolated NK cells were used for the evaluation of NK cell cytotoxicity in normal and immunodeficient mice in vitro or after adoptive transfer in vivo Isolation of NK cells to study their role in experimental mouse models of immune mediated liver injury or chronic infection with parasitic helminths Miltenyi Biotec Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 CD49b DX5 MicroBeads Mouse Order No 130 052 501 1 3 Reagent and instrument requirements Buffer degassed Prepare a solution containing PBS phosphate buffered saline pH 7 2 0 5 BSA bovine serum albumin and 2 mM EDTA by diluting MACS BSA Stock Solution 130 091 376 1 20 in autoMACS Rinsing Solution 130 091 222 Keep buffer cold 4 8 C A Note EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula A ACD A or citrate phospha
7. ove cell clumps www miltenyibiotec com A Miltenyi Biotec Inc 2303 Lindbergh Street Auburn CA 95602 USA Phone 800 FOR MACS 530 888 8871 Fax 530 888 8925 page 1 3 0 0 L 000 0rL 2 Protocol 2 1 Sample preparation Prepare a single cell suspension from lymphoid organs non lymphoid tissue or peripheral blood using standard methods see General Protocols in the User Manuals or visit www miltenyibiotec com A Dead cells may bind non specifically to MACS MicroBeads In case of high numbers of dead cells we recommend to remove dead cells by density gradient centrifugation or using the Dead Cell Removal Kit 130 090 101 an b 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 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 1 Determine cell number 2 Centri
8. sing with appropriate amount of buffer MS 500 uL LS 3 mL 3 Apply cell suspension onto the column 4 Collect unlabeled cells which pass through and wash column with appropriate amount of buffer Perform washing steps by adding buffer three times each timeonce the column reservoir is empty MS 3x500 uL LS 3x3 mL Collect total effluent This is the unlabeled cell fraction 5 Remove column from the separator and place it on a suitable collection tube 6 Pipette appropriate amount of buffer onto the column Immediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column MS 1 mL LS 5 mL A Note To increase the purity of the magnetically labeled fraction it can be passed over a new freshly prepared 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 see LD Column data sheet 2 Prepare column by rinsing with 2 mL of buffer 3 Apply cell suspension onto the column 4 Collect unlabeled cells which pass through and wash column with 2x1 mL of buffer Collect total effluent This is the unlabeled cell fraction Depletion with CS Columns 1 Assemble CS Column and place it in the magnetic field of a suitable MACS Separator see CS Column data sheet 2 Prepare colum
9. te dextrose CPD BSA can be replaced by other proteins such as gelatine mouse serum or fetal calf serum Buffers or media containing Ca or Mg are not recommended for use MACS Columns and MACS Separators CD49b cells can be enriched by using MS LS or XS Columns positive selection CD49b DX5 MicroBeads can be used for depletion of CD49b cells on LD CS or D Columns Cells which strongly express CD49b can also be depleted using MS LS or XS Columns Positive selection or depletion can also be performed by using the autoMACS Separator Column max number max number Separator of labeled cells of total cells Positive selection MS 10 2x108 MiniMACS OctoMACS VarioMACS SuperMACS LS 108 2x10 MidiMACS QuadroMACS VarioMACS SuperMACS XS 10 2x1010 SuperMACS Depletion LD 108 5x108 MidiMACS QuadroMACS VarioMACS SuperMACS CS 2x108 VarioMACS SuperMACS D 10 SuperMACS Positive selection or depletion autoMACS 2x108 4x10 autoMACS A Note Column adapters are required to insert certain columns into VarioMACS Separator or SuperMACS Separator For details see MACS Separator data sheets Optional Fluorochrome conjugated CD49b antibodies eg CD49b DX5 FITC 130 091 814 CD49b DX5 PE 130 091 816 or CD49b DX5 APC 130 091 813 Optional PI propidium iodide or 7 AAD for flow cytometric exclusion of dead cells Optional Pre Separation Filters 130 041 407 to rem
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