CD4+CD25+ Regulatory T Cell Isolation Kit
Contents
1. below are for an initial starting cell number of up to 107 leukocytes For larger initial cell numbers scale up volumes accordingly 1 Centrifuge isolated CD4 T cells at 300xg for 10 minutes Aspirate supernatant completely 2 Resuspend cell pellet in 90 uL of buffer 3 Add 10 uL of Anti PE MicroBeads 4 Mix well and refrigerate for 15 minutes in the dark 4 8 C A Note Working on ice may require increased incubation times Higher temperatures and or longer incubation times may lead to non specific cell labeling 5 Wash cells by adding 1 2 mL of buffer and centrifuge at 300xg for 10 minutes Aspirate supernatant completely 6 Resuspend up to 108 cells in 500 uL of buffer A Note For larger cell numbers scale up buffer volume accordingly 7 Proceed to magnetic separation 2 5 Miltenyi Biotec Unless otherwise specifically indicated all Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use Order no 130 091 041 2 5 Magnetic separation Positive selection of CD4 CD25 regulatory T cells mm Positive selection with MS Columns A To achieve highest purities always perform two consecutive column runs 1 Place MS Column in the magnetic field of a suitable MACS Separator For details see MS Column data sheet 2 Prepare column by rinsing with 500 uL of buffer 3 Apply cell suspension onto the column 4 Let cells pass through and wash column with 32. expiration date is indicated on the vial label Miltenyi Biotec Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 CD4 CD25 Regulatory T Cell Isolation Kit mouse Order no 130 091 041 1 1 Principle of MACS Separation Mouse CD4 CD25 regulatory T cells are isolated in a two step procedure First CD4 T cells are pre enriched by depletion of unwanted cells Then CD25 cells are positively selected from the enriched CD4 T cell fraction For the isolation of CD4 T cells non CD4 T cells are indirectly magnetically labeled with a cocktail of biotin conjugated antibodies and Anti Biotin MicroBeads In parallel the cells are labeled with CD25 PE The cell suspension is loaded onto a MACS Column which is placed in the magnetic field of a MACS Separator The magnetically labeled non CD4 T cells are retained in the column while the unlabeled CD4 T cells run through For the isolation of CD4 CD25 cells the CD25 PE labeled cells in the enriched CD4 T cell fraction are magnetically labeled with Anti PE MicroBeads The cell suspension is again loaded onto a column which is placed in the magnetic field of a MACS Separator The magnetically labeled CD4 CD25 cells are retained in the column while the unlabeled cells run through After removal of the column from the magnetic field the retained CD4 CD25 cells are eluted as the positively selected cell fra
3. no warranties expressed or implied which extend beyond the technical specifications of the products Miltenyi Biotec GmbH 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 autoMACS MidiMACS MiniMACS OctoMACS QuadroMACS SuperMACS and VarioMACS are trademarks of Miltenyi Biotec GmbH 2006 Miltenyi Biotec GmbH Printed in Germany www miltenyibiotec com page 4 4
4. 4 T cells as well as CD25 PE and Anti PE MicroBeads for subsequent positive selection of CD4 CD25 regulatory T cells Example of applications Isolation of CD4 CD25 regulatory T cells from single cell suspensions of spleen or lymph nodes for e co culture experiments with DCs to study priming of DCs for tolerance induction in vitro and after adoptive transfer of primed DCs in vivo e adoptive transfer experiments e g from UV exposed mice to analyze the role of regulatory T cells during induction and elicitation of hapten specific tolerance 1 3 Reagent and instrument requirements Buffer Phosphate buffered saline PBS pH 7 2 supplemented with 0 5 bovine serum albumin BSA and 2 mM EDTA Keep buffer cold 4 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 gelatine murine serum or fetal calf serum Buffers or media containing Ca or Mg are not recommended for use Optional Pre Separation Filters 130 041 407 to remove cell clumps Optional Propidium iodide PI or 7 AAD for the exclusion of dead cells Optional Fluorochrome conjugated CD4 antibody e g CD4 FITC 130 091 608 CD4 PE 130 091 607 or CD4 APC 130 091 611 MACS Columns and MACS Separators Depletion of
5. 90 L08 000 0rL 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 2 2 Sample preparation Magnetic labeling of non CD4 T cells and fluorescent labeling of CD25 cells 2 3 2 4 Magnetic separation Depletion of non CD4 T cells Magnetic labeling of CD25 cells 2 5 Magnetic separation Positive selection of CD4 CD25 regulatory T cells 3 Example of a separation using the CD4 CD25 Regulatory T Cell Isolation Kit 4 References 1 Description Components 1 mL CD4 CD25 Regulatory T Cell Biotin Antibody Cocktail mouse Cocktail of biotin conjugated monoclonal anti mouse antibodies against CD8a Ly 2 isotype rat IgG2a CD11b Mac 1 isotype rat IgG2b CD45R B220 isotype rat IgG2a CD49b DX5 isotype rat IgM and Ter 119 isotype rat IgG2b 2 mL Anti Biotin MicroBeads MicroBeads conjugated to monoclonal anti biotin antibody isotype mouse IgG1 1 mL CD25 PE mouse Monoclonal anti mouse CD25 antibody conjugated to R Phycoerythrin PE clone 7D4 isotype rat IgM 1 mL Anti PE MicroBeads MicroBeads conjugated to monoclonal anti PE antibodies isotype mouse IgG1 Size For 10 leukocytes up to 100 separations Product format All components are supplied in buffer containing stabilizer and 0 05 sodium azide Storage Store protected from light at 2 8 C Do not freeze The
6. ction and once again separated over a new column to achieve highest purities Cell suspension Depletion of non CD4 T cells 1 Indirect magnetic labeling of non CD4 T cells with Biotin Antibody Cocktail and Anti Biotin MicroBeads 2 Labeling of CD25 cells with CD25 PE antibody 3 Magnetic separation using LD Column or autoMACS Flow through fraction CD4 T cells Positive selection of CD4tCD25 cells 1 Indirect magnetic labeling of CD25 cells with Anti PE MicroBeads 2 Magnetic separation using two MS Columns or autoMACS Elution from column CD4 CD25 regulatory T cells 1 2 Background and product applications The CD4 CD25 Regulatory T Cell Isolation Kit was developed for the isolation of mouse CD4 CD25 regulatory T cells from single cell suspensions of spleen or lymph nodes CD4 CD25 immunoregulatory T cells have been shown to actively suppress immune responses against autologous and foreign antigens 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 4 90 L08 000 0rL in vivo and in vitro CD25 the IL 2Ra chain is also expressed on activated CD8 T cells dendritic cells DCs and B cells The kit contains a cocktail of lineage specific biotin conjugated antibodies against CD8 Ly 2 CD11b Mac 1 CD45R B220 CD49b DX5 Ter 119 and Anti Biotin MicroBeads for depletion of non CD
7. d cell pellet in buffer Depletion with LD Column 500 uL for up to 1 25x108 cells Depletion with autoMACS 500 uL for up to 10 cells A Note For larger cell numbers scale up buffer volume accordingly 10 Proceed to magnetic separation 2 3 www miltenyibiotec com A page 2 4 90 L08 000 0rL nm 2 3 Magnetic separation Depletion of non CD4 T cells Depletion with LD Column 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 Perform washing steps by adding buffer successively once the column reservoir is empty Collect total effluent This is the unlabeled CD4 T cell fraction 5 Proceed to 2 4 for the enrichment of CD4 CD25 T cells Depletion with the autoMACS Separator A Refer to the auttoMACS User Manual for instructions on how to use the autoMACS Separator 1 Prepare and prime the autoMACS Separator 2 Place the tube containing the magnetically labeled cells in the autoMACS Separator Choose separation program Depl025 3 Collect the unlabeled fraction from outlet port neg1 This is the enriched CD4 T cell fraction 4 Proceed to 2 4 for the enrichment of CD4 CD25 T cells Lot p 2 4 Magnetic labeling of CD25 cells A Volumes for magnetic labeling given
8. g of antibodies on the cell surface and non specific cell labeling A Volumes for magnetic labeling given below are for a starting cell number of 107 leukocytes 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 leukocytes use twice the volume of all indicated reagent volumes and total volumes A For an 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 number of leukocytes 2 Centrifuge cells at 300xg for 10 minutes Aspirate supernatant completely 3 Resuspend cell pellet in 40 uL of buffer per 107 total cells 4 Add 10 uL of Biotin Antibody Cocktail per 107 total cells 5 Mix well and refrigerate for 10 minutes 4 8 C A Note Working on ice may require increased incubation times Higher temperatures and or longer incubation times may lead to non specific cell labeling 6 Add 30 uL of buffer 20 uL of Anti Biotin MicroBeads and 10 uL of CD25 PE antibody per 107 total cells 7 Mix well and refrigerate for an additional 15 minutes in the dark 4 8 C 8 Wash cells by adding 1 2 mL of buffer per 107 total cells and centrifuge at 300xg for 10 minutes Aspirate supernatant completely 9 Resuspen
9. non CD4 T cells is performed on an LD Column The subsequent positive selection of CD4 CD25 T cells is performed on two MS Columns Depletion and positive selection can also be performed by using the autoMACS Separator Column Max numberof Max numberof Separator labeled leukocytes total leukocytes Depletion LD 108 5x108 MidiMACS QuadroMACs VarioMACS SuperMACS Positive selection MS 107 2x108 MiniMACS OctoMACS VarioMACS SuperMACS Depletion and positive selection autoMACS 2x108 4x10 autoMACS 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 Unless otherwise specifically indicated all Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use Order no 130 091 041 2 Protocol 2 1 Sample preparation Prepare a single cell suspension from spleen or lymph nodes using standard methods A Note The Kit is not optimized for the isolation of regulatory T cells from blood and thymus A Red blood cell lysis or density gradient centrifugation is not necessary since the CD4 CD25 T Cell Biotin Antibody Cocktail contains Anti Ter 119 antibody 2 2 Magnetic labeling of non CD4 cells and fluorescent labeling of CD25 cells nN A Work fast keep cells cold and use pre cooled solutions This will prevent cappin
10. rom analysis based on scatter signals and PI fluorescence Spleen cells before separation CD25 PE CD4 FITC CD4 T cells after depletion of non CD4 T cells CD25 PE CD4 FITC Isolated CD4 CD25 regulatory T cells CD25 PE CD4 FITC Miltenyi Biotec Unless otherwise specifically indicated all Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use Order no 130 091 041 4 References 1 Fallarino F et al 2003 Modulation of tryptophan catabolism by regulatory T cells Nat Immunol 4 12 1206 1212 3982 Schwarz A et al 2004 Ultraviolet Radiation Induced Regulatory T Cells Not Only Inhibit the Induction but Can Suppress the Effector Phase of Contact Hypersensitivity J Immunol 172 1036 1043 3791 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
11. x500 uL of buffer Perform washing steps by adding buffer three times once the column reservoir is empty 5 Remove column from the separator and place it on a suitable collection tube 6 Pipette 1 mL of buffer onto the column Immediately flush out the magnetically labeled cells CD4 CD25 cells by firmly pushing the plunger into the column A Note To perform a second column run you may elute the cells directly from the first onto the second equilibrated column instead of a collection tube 7 Repeat the magnetic separation procedure as described in steps 1 6 by using a new MS Column Positive selection with the autoMACS Separator A Refer to the autoMACS User Manual for instructions on how to use the autoMACS Separator 1 Prepare and prime the autoMACS Separator 2 Place the tube containing the magnetically labeled cells in the autoMACS Separator Choose separation program Posseld2 3 Collect the positive fraction from outlet port pos2 This is the enriched CD4 CD25 T cell fraction www miltenyibiotec com A page 3 4 90 L08 000 0rL 3 Example of a separation using the CD4 CD25 Regulatory T Cell Isolation Kit CD4 CD25 regulatory T cells were isolated from mouse spleen cell suspension using the CD4 CD25 Regulatory T Cell Isolation Kit an LD and two MS Columns a MidiMACS Separator and a MiniMACS Separator Cells were additionally stained with CD4 FITC Cell debris and dead cells were excluded f
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