CD45RO MicroBeads
Contents
1. 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 CD45RO cells can be enriched by using MS LS or XS Columns positive selection CD45RO MicroBeads can be used for depletion of CD45ROt cells on LD CS or D Columns Cells which strongly express the CD45RO antigen can also be depleted using MS LS or XS Columns CD45RO MicroBeads are less suitable for depletion or enrichment of monocytes macrophages and granulocytes Positive selection or depletion can also be performed by using the auttoMACS Separator Column max number max number Separator of labeled cells of total cells Positive selection MS 10 2x10 MiniMACS OctoMACS VarioMACS SuperMACS LS 108 2x10 MidiMACS QuadroMACS VarioMACS SuperMACS XS 10 2x109 SuperMACS Depletion LD 108 5x108 MidiMACS QuadroMACS VarioMACS SuperMACS CS 2x108 VarioMACS SuperMACS D 10 SuperMACS Positive selection or depletion autoMACS 2x10 4x10 autoM ACS 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 Miltenyi Biotec GmbH Friedrich Ebert St2. 2595 2601 2490 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 autoMACS and MACS are registered trademarks and MidiMACS MiniMACS OctoMACS QuadroMACS SuperMACS and VarioMACS are trademarks of Miltenyi Biotec GmbH Ficoll Paque is a trademark of GE Healthcare companies Copyright 2012 Miltenyi Biotec GmbH All rights reserved Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 3 3
3. Cell Removal Kit 130 090 101 O 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 A If CD45RO MicroBeads are used for positive selection of CD45RO T helper or cytotoxic suppressor cells in combination with CD4 or CD8 MultiSort Kit we recommend using a 1 20 dilution instead of 1 5 for the magnetic labeling This increases the purity of the CD4 CD45RO and CD8 CD45RO cells respectively but may decrease the recovery 1 Determine cell number 2 Centrifuge at 300xg for 10 minutes Aspirate supernatant completely 3 Resuspend cell pellet in 80 uL of buffer per 107 total cells 4 Add 20 uL of CD45RO MicroBeads per 10 total cells 5 Mix well and refrigerate for 15 minutes 4 8 C A Note Working on ice may require in
4. D8 T cell subsets and in a lower amount on monocytes macrophages and granulocytes The CD45RO antibody recognizes a 180 kDa isoform of the leukocyte common antigen LCA CD45RO MicroBeads can be used for the positive selection or depletion of CD45RO cells For isolation of CD45RO T cell subsets the CD45RO MicroBeads can be combined with MACS CD4 and CD8 T Cell Isolation Kits or CD4 and CD8 MultiSort Kits Cells isolated with CD45RO MicroBeads can be used for various studies such as cytokine expression or receptor signaling CD45RO MicroBeads human Order no 130 046 001 Examples of applications Depletion of human CD45RO cells from peripheral blood or lymphoid tissue for further selection of CD45RO naive T cell subsets Positive selection or depletion of human CD45RO memory T cells from preselected CD4 or CD8 cells isolated from peripheral blood using MACS Cell Isolation Kits or from lymphoid tissue using MACS MultiSort Kits Isolation of human CD45RA naive T cells from presorted CD4 or CD8 cells by depletion of CD45RO cells 1 3 Reagent and instrument requirements Buffer Prepare a solution containing phosphate buffered saline PBS pH 7 2 0 5 bovine serum albumin 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 Degas buffer before use as air bubbles could block the column
5. Z0 6 0 000 07L 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 Examples of separations using CD45RO MicroBeads 4 References 1 Description Components 2 mL CD45RO MicroBeads human MicroBeads conjugated to monoclonal mouse anti human CD45RO_ antibodies isotype mouse IgG2a Size For 10 total cells up to 100 separations Product format CD45RO 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 CD45RO cells are magnetically labeled with CD45RO MicroBeads Then the cell suspension is loaded on a MACS Column which is placed in the magnetic field of a MACS Separator The magnetically labeled CD45RO cells are retained on the column The unlabeled cells run through this cell fraction is depleted of CD45RO cells After removal of the column from the magnetic field the magnetically retained CD45RO cells can be eluted as the positively selected cell fraction 1 2 Background and product applications CD45RO MicroBeads were developed for direct magnetic labeling of lymphocyte subsets CD45RO is brightly expressed on CD4 and C
6. creased 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 CD4 FITC Order no 130 046 001 130 080 501 and refrigerate for 5 minutes 4 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 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 nn 2 3 Magnetic separation A Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD45RO cells For details 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 For details refer to the respective MACS Column data sheet 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 cells that pass through and wash column with appropriate amount of buffer Perform washing steps by adding buffer three times Only add new buffer when 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 s
7. ed with CD45RO FITC B A PBMCs before separation CD45RO cells Relative cell number Relative cell number CD45RO FITC CD45RO FITC D PBMCs before separation CD45RO cells Relative cell number Relative cell number CD45RO FITC CD45RO FITC Order no 130 046 001 4 References 1 Krug A et al 2001 Toll like receptor reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL 12 Eur J Immunol 31 3026 3037 1215 2 Coccia EM et al 1999 Interleukin 12 Induces Expression of Interferon Regulatory Factor 1 via Signal Transducer and Activator of Transcription 4 in Human T Helper Type 1 Cells J Biol Chem 274 6698 6703 680 3 Wolthers K C et al 1999 Normal T Cell Telomerase Activity and Upregulation in Human Immunodeficiency Virus 1 Infection Blood 93 1011 1019 570 4 Jonuleit H et al 2001 Identification and Functional Characterisation of Human CD4 CD25 T Cells with Regulatory Properties Isolated from Peripheral Blood J Exp Med 193 1285 1294 1038 5 Kimmig S et al 2002 Two Subsets of Naive T Helper Cells with Distinct T Cell Receptor Excision Circle Content in Human Adult Peripheral Blood J Exp Med 195 789 794 1294 6 Malmberg K J et al 2001 Inhibition of Activated Memory CD45RO T Cells by Oxidative Stress Associated with Block of NF B Activation J Immunol 167
8. eparator and place it on a suitable collection tube 6 Pipette an 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 A Note To increase the purity of the magnetically labeled fraction pass the cells 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 For details 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 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 Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 2 3 Z0 6 0 000 0rL 3 Apply cell suspension onto the column 4 Collect unlabeled cells which pa
9. r 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 Miltenyi Biotec Inc 2303 Lindbergh Street Auburn CA 95602 USA Phone 800 FOR MACS 530 888 8871 Fax 530 888 8925 page 1 3 Z0 6 0 000 01L Optional Fluorochrome conjugated CD45RO antibody additional fluorochrome conjugated staining antibodies e g CD4 FITC 130 080 501 CD8 APC 130 091 076 CD25 PE 130 091 024 Optional PI propidium iodide or 7 AAD for the flow cytometric exclusion of dead cells Optional Pre Separation Filters 130 041 407 to remove dead cells 2 Protocol When working with anticoagulated peripheral blood or buffy coat peripheral blood mononuclear cells PBMCs should be isolated by density gradient centrifugation e g using Ficoll Paque For details see section General Protocols in the User Manuals or visit 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 prepare a single cell suspension by a standard preparation method For details see section General Protocols in the User Manuals or visit www miltenyibiotec 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
10. ss through and wash column with 30 mL buffer from 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 Magnetic separation with the autoMACS Separator A Refer to the auttoMACS 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 one of the 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 section autoMACS Cell Separation Programs 3 When using the program Possel collect positive fraction from outlet port posl This is the purified CD45RO cell fraction When using the program Depletes collect unlabeled fraction from outlet port neg1 This is the CD45RO cell fraction 3 Examples of separations using CD45RO MicroBeads CD45RO cells were separated from PBMCs using CD45RO MicroBeads For positive selection CD45RO cells were isolated using an MS Column and a MiniMACS Separator A For depletion of CD45RO cells the sample was separated over an LD Column in a MidiMACS Separator Cells are fluorescently stain
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