CD3 MicroBeads - Miltenyi Biotec
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1. 0 990 000 0rL Miltenyi Biotec 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 3 Example of a separation using CD3 MicroBeads 4 References 1 Description Components 2 mL CD3 MicroBeads human MicroBeads conjugated to monoclonal anti human CD3 antibodies isotype mouse IgG2a Size For 10 total cells up to 100 separations Product format CD3 MicroBeads are supplied as a suspension containing stabilizer and 0 05 sodium azide Store protected from light at 4 8 C Do not freeze The expiration date is indicated on the vial label Storage 1 1 Principle of MACS separation First the CD3 cells are magnetically labeled with CD3 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 CD3 cells are retained on the column The unlabeled cells run through and this cell fraction is depleted of CD3 cells After removal of the column from the magnetic field the magnetically retained CD3 cells can be eluted as the positively selected cell fraction 1 2 Background and product applications CD3 is expressed on all T cells and is associated with the T cell receptor 70 80 of human peripheral blood lymphocytes and 65 85 of thymocytes are CD3 The epit2. Paque see General Protocols in the User Manuals or visit www miltenyibiotec com protocols A Note Remove platelets after density gradient separation resuspend cell pellet in buffer and centrifuge at 200xg for 10 15 minutes at 20 C Carefully remove supernatant Repeat washing step and carefully remove supernatant When working with tissues prepare a single cell suspension by a standard preparation method see General Protocols in the User Manuals or visit www iniltenyibiotec com protocols A Note 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 ae b 2 2 Magnetic labeling A Work fast keep the 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 107 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 2x107 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 colum
3. Beads and a MiniMACS Separator with an MS Column The cells are fluorescently stained with CD3 FITC 130 080 401 Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence PBMCs before separation a 2 E 5 Z T v v 2 FEN Aay FAN 3 A CD3 FITC CD3 cells o 2 5 E T v v 2 oO T cc CD3 FITC Order No 130 050 101 4 References f Pitti RM Marsters SA Lawrence DA Roy M Kischkel FC Dowd P Huang A Donahue CJ Sherwood SW Baldwin DT Godowski PJ Wood WI Gurney AL Hillan KJ Cohen RL Goddard AD Botstein D Ashkenazi A 1998 Genomic Amplification of a Decoy Receptor for Fas Ligand in Lung and Colon Cancer Nature 396 699 703 517 2 Heath S Tew J Szakal A Burton G 1995 Follicular Dendritic Cells and Human Immunodeficiency Virus Infectivity Nature 377 740 744 177 3 Lorenzen DR D x F W lk U Tsirpouchtsidis A Haas G Meyer TF 1999 Immunoglobulin A1 Protease an Exoenzyme of Pathogenic Neisseriae Is a Potent Inducer of Proinflammatory Cytokines J Exp Med 190 1049 1058 882 4 Heidenreich F Jovin T 1996 Synthesis of anti acetylcholine receptor antibodies by CD5 B cells from peripheral blood of myasthenia gravis patients J Neurol 243 57 62 441 5 Klein U Kiippers R Rajewsky K 1994 Variable Region Gene Analysis of B Cell Subsets Derived from a 4
4. ells which pass through and wash column with appropriate amount of buffer Perform washing steps by adding buffer three times each time once 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 column 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 C
5. mber Separator of labeled cells of total cells Positive selection MS 107 2x108 MiniMACS OctoMACS VarioMACS SuperMACS LS 108 2x10 MidiMACS QuadroMACS VarioMACS SuperMACS XS 10 2x10 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 CD3 antibody for flow cytometric analysis e g CD3 FITC 130 080 401 CD3 PE 130 091 374 or CD3 APC 130 091 373 Optional PI propidium iodide or 7 AAD for flow cytometric exclusion of dead cells Optional Pre Separation Filters 130 041 407 to remove cell clumps 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 3 0 990 000 0rL 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 e g Ficoll
6. n 1 Determine cell number 2 Centrifuge cell suspension at 300xg for 10 minutes Pipette off supernatant completely 3 Resuspend cell pellet in 80 uL of buffer per 10 total cells 4 Add 20 uL of CD3 MicroBeads per 107 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 staining antibodies e g add 10 uL of CD3 FITC 130 080 401 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 up to 1 25x108 cells in 500 uL of buffer 9 Proceed to magnetic separation 2 3 Order No 130 050 101 zm 2 3 Magnetic separation A Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD3 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 rinsing with appropriate amount of buffer MS 500 uL LS 3 mL 3 Apply cell suspension onto the column 4 Collect unlabeled c
7. olumn 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 Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 2 3 0 990 000 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 autoMACS 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 CD3 cell fraction When using the program Depletes collect unlabeled fraction outlet port neg1 This is the CD3 cell fraction 3 Example ofa separation using CD3 MicroBeads Separation of PBMCs using CD3 Micro
8. ope recognized by CD3 MicroBeads is located on the CD3 chain Examples of applications e CD3 MicroBeads were used for the positive selection or depletion of T cells from peripheral blood bronchial lavage cell culture or various tissues such as lymphoid nasal and tumor tissue T cells isolated by MACS Technology have been used for various studies e g on T cell cytotoxicity T cell activation HIV infectivity signal transduction and surface marker expression CD3 MicroBeads human Order No 130 050 101 1 3 Reagent and instrument requirements e Buffer degassed Prepare a solution containing PBS phosphate buffered saline pH 7 2 0 5 BSA and 2 mM EDTA by diluting MACS BSA Stock Solution 130 091 376 1 20 with 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 phosphate dextrose CPD BSA can be replaced by other proteins such as human serum albumin human serum or fetal calf serum Buffers or media containing Ca or Mg are not recommended for use MACS Columns and MACS Separators CD3 cells can be enriched by using MS LS or XS Columns positive selection CD3 MicroBeads can be used for depletion of CD3 cells on LD CS or D Columns Positive selection or depletion can also be performed by using the autoMACS Separator Column max number max nu
9. year old Child Somatically Mutated Memory B Cells Accumulate in the Periperal Blood Already at Young Age J Exp Med 180 1383 1393 75 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 Ficoll Paque is a trademark of GE Healthcare companies MACS is a registered trademark of Miltenyi Biotec GmbH Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 3 3
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