CD4+ CD62L+ T Cell Isolation Kit II
Contents
1. 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 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 mouse serum or fetal calf serum Buffers or media containing Ca or Mg are not recommended for use MACS Columns and MACS Separators Depletion of non CD4 T cells is performed on an LS Column The subsequent positive selection of CD4 CD62L T cells is performed on an MS Column Depletion and positive selection can also be performed by using the autoMACS Separator Column Max numberof Max numberof Separator labeled leukocytes total leukocytes Depletion LS 108 2x10 MidiMACS QuadroMACS VarioMACS SuperMACS Positive selection MS 10 2x108 MiniMACS OctoMACS VarioMACS SuperMACS Depletion and positive selection autoM ACS 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 Optional Fluorochrome conjugated antibodies for flow cytometric2. a MidiMACS and a MiniMACS Separator The cells were fluorescently stained with CD4 FITC 130 091 608 and CD62L APC 130 091 805 for detection of naive T cells a and with CD62L APC and CD44 PE for detection of central memory T cells b Additionally cells were stained with CD25 PE 130 091 013 to illustrate the removal of CD25 CD62L regulatory T cells c Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence Before separation a b U U a a q _l l N N O O la la U U CD4 FITC CD44 PE Pre enriched CD4 T cells after depletion of non CD4 T cells a a 3 U U CD4 FITC CD44 PE Isolated CD4 CD62L T cells U U Q Q T E l l N N O O a lal U U CD4 FITC CD44 PE Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 3 4 cO CL L c00 O0F L c Before separation CD62L APC CD25 PE Pre enriched CD4 T cells after depletion of non CD4 T cells CD62L APC CD25 PE Isolated CD4 tCD62L T cells CD62L APC CD25 PE Order no 130 093 227 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 ex
3. distinguished from naive T helper cells by their high expression of CD44 Furthermore CD62L is expressed on most thymocytes naive CD8 T cells B cells dendritic cells macrophages NK cells neutrophils eosinophils regulatory T cells and TCRy d T cells For isolation of CD4 CD62L T helper cells the non T helper cells as well as regulatory T cells and TCRy T cells are depleted by indirect magnetic labeling using a cocktail of lineage specific biotin conjugated antibodies against CD8a Ly 2 CD45R B220 CD49b DX5 CD11b Mac 1 and Ter 119 as well as antibodies against Miltenyi Biotec GmbH Friedrich Ebert StraRe 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 cO CL L c00 O0V L CD25 and TCRy 6 in combination with Anti Biotin MicroBeads Subsequently CD4 CD62L T cells are positively selected from the enriched CD4 T helper cell fraction with CD62L L selectin MicroBeads Example applications Isolation of CD4 CD62L T cells from single cell suspensions of spleen and lymph nodes for analysis of T cell activation by antigen presenting cells studies on cytokine expression and receptor signaling adoptive transfer experiments 1 3 Reagent and instrument requirements
4. labeled with a cocktail of biotin conjugated antibodies and Anti Biotin MicroBeads The labeled cells are subsequently depleted by separation over a MACS Column In the second step CD4 CD62L T cells are directly labeled with CD62L L selectin MicroBeads and isolated by positive selection from the pre enriched CD4 T cell fraction The magnetically labeled CD4 CD62L T cells are retained on the column and eluted after removal of the column from the magnetic field Single cell suspension Depletion of non CD4 T cells 1 Indirect magnetic labeling of non CD4 T cells with the CD4 T Cell Biotin Antibody Cocktail II and Anti Biotin MicroBeads 2 Magnetic separation using an LS Column or the autoMACS Separator program Depletes Flow through fraction pre enriched CD4 T cells Positive selection of CD4 CD62L T cells 1 Direct magnetic labeling of CD4 CD62L T cells with CD62L L selectin MicroBeads 2 Magnetic separation using an MS Column or the autoMACS Separator program Possel Elution from column CD4 CD62L T cells 1 2 Background and product applications The CD4 CD62L T Cell Isolation Kit II has been developed for the isolation of CD4 CD62L T helper cells from spleen and lymph nodes CD62L L selectin is highly expressed on naive T cells and down regulated upon activation It is also expressed on a small subset of memory T helper cells the central memory T cells which can be
5. 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 the number of leukocytes 2 Centrifuge cells at 300xg for 10 minutes Aspirate supernatant completely 3 Resuspend cell pellet in 400 uL of buffer per 10 total cells 4 Add 100 uL of CD4 T Cell Biotin Antibody Cocktail II per 108 total cells 5 Mix well and refrigerate for 10 minutes 4 8 C 6 Add 300 uL of buffer and 200 uL of Anti Biotin MicroBeads per 108 total cells 7 Mix well and refrigerate for 15 minutes 4 8 C 8 Wash cells by adding 10 mL of buffer and centrifuge at 300xg for 10 minutes at 4 8 C Aspirate supernatant completely 9 Resuspend up to 108 cells in 500 uL of buffer A Note For higher cell numbers scale up buffer volume accordingly 10 Proceed to magnetic separation 2 3 2 3 Magnetic separation Depletion of non CD4 T cells m Depletion with LS Column 1 Place LS Column in the magnetic field of a suitable MACS Separator For details see LS Column data sheet 2 Prepare column by rinsing with 3 mL of buffer 3 Apply cell suspension onto the column 4 Collect unlabeled cells which pass through and wash column with 3x3 mL of buffer Perform washing steps by adding bu
6. 6 Resuspend up to 10 cells in 500 uL of buffer 7 Proceed to magnetic separation 2 5 2 5 Magnetic separation Positive selection of CD4 CD62L T cells T Positive selection with MS Columns 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 Wash column with 3x500 uL of buffer Perform washing steps by adding buffer three times Only add new buffer when 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 CD4 CD62L T cells by firmly pushing the plunger into the column Order no 130 093 227 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 autoM ACS Separator Choose the separation program Possel 3 Collect the positive fraction from outlet port posl This is the enriched CD4 CD62L T cell fraction 3 Example of a separation using the CD4 CD62L T Cell Isolation Kit II CD4 CD62L T cells were isolated from a mouse spleen cell suspension using the CD4 CD62L T Cell Isolation Kit II an LS and an MS Column
7. analysis e g CD4 FITC 130 091 608 CD4 PE 130 091 607 CD4 APC 130 091 611 CD62L PE 130 091 794 CD62L APC 130 091 805 CD25 PE 130 091 013 and CD44 antibodies Optional Propidium iodide PI or 7 AAD for flow cytometric exclusion of dead cells Optional Pre Separation Filters 130 041 407 to remove cell clumps 2 Protocol 2 1 Sample preparation Prepare a single cell suspension from spleen and lymph nodes using standard methods A Note Red blood cell lysis or density gradient centrifugation is not necessary since red blood cells are depleted in the first magnetic separation step on the basis of expression of Ter 119 a red blood cell restricted surface antigen of the mouse Order no 130 093 227 A Note Dead cells may bind non specifically to MACS MicroBeads In case of high numbers of dead cells removal of dead cells by density gradient centrifugation or the Dead Cell Removal Kit 130 090 101 is recommended 5 2 2 Magnetic labeling of non CD4 T cells D 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 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 2x108 leukocytes use twice
8. cO Z L L c00 O0F L 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 2 2 2 3 Sample preparation Magnetic labeling of non CD4 T cells Magnetic separation Depletion of non CD4 T cells 2 4 Magnetic labeling of CD4 CD62L T cells 2 5 Magnetic separation Positive selection of CD4 CD62L T cells 3 Example of a separation using the CD4 CD62L T Cell Isolation Kit II 1 Description Components 1 mL CD4 T Cell Biotin Antibody Cocktail II mouse Cocktail of biotin conjugated monoclonal anti mouse antibodies against CD8a CD45R CD11b CD25 CD49b TCRy 6 and Ter 119 2 mL Anti Biotin MicroBeads MicroBeads conjugated to monoclonal anti biotin antibody isotype mouse IgG1 2 mL CD62L L selectin MicroBeads MicroBeads conjugated to monoclonal anti mouse CD62L L selectin isotype rat IgG2a antibody Size For 1x10 total cells 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 expiration date is indicated on the vial label CD4 CD62L T Cell Isolation Kit II mouse Order no 130 093 227 1 1 Principle of MACS Separation The isolation of mouse CD4 CD62L T cells is performed in a two step procedure First non CD4 T cells are indirectly magnetically
9. ffer three times Only add new buffer when the column reservoir is empty Collect total effluent This contains the unlabeled pre enriched CD4 T cell fraction 5 Proceed to 2 4 for the isolation of CD4 CD62L T cells Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 2 4 cO CL L c00 O0F L Depletion 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 auttoMACS Separator 2 Place the tube containing the magnetically labeled cells in the autoMACS Separator Choose the separation program Depletes 3 Collect the unlabeled fraction from outlet port neg1 This is the pre enriched CD4 T cell fraction 4 Proceed to 2 4 for the isolation of CD4 CD62L T cells e b 2 4 Magnetic labeling of naive CD4 T cells A Volumes for magnetic labeling given below are for an initial starting cell number of up to 108 leukocytes For larger initial cell numbers scale up volumes accordingly 1 Centrifuge the cells at 300xg for 10 minutes Aspirate supernatant completely Resuspend cell pellet in 800 uL of buffer Add 200 uL of CD62L L selectin MicroBeads Mix well and refrigerate for 15 minutes 4 8 C 5 Wash cells by adding 10 mL of buffer and centrifuge at 300xg for 10 minutes Aspirate supernatant completely
10. plosive 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 autoMACS MidiMACS MiniMACS OctoMACS QuadroMACS SuperMACS and VarioMACS are trademarks of Miltenyi Biotec GmbH 2007 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 4 4
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