CD8+ Dendritic Cell Isolation Kit
Contents
1. 0 788 000 0F L Contents 1 Description 1 1 Principle of the MACS Separation 1 2 Background information 1 3 1 4 Applications Reagent and instrument requirements 2 Protocol 2 1 Sample preparation 2 2 Magnetic labeling of T B and NK cells 2 3 Magnetic separation Depletion of T B and NK cells 2 4 Magnetic labeling of CD8 dendritic cells 2 5 Magnetic separation Positive selection of CD8 dendritic cells 3 Example of a separation using the CD8 Dendritic Cell Isolation Kit 4 References 1 Description Components 1 mL CD8 Dendritic Cell Biotin Antibody Cocktail mouse Cocktail of biotin conjugated monoclonal anti mouse antibodies against CD90 2 Thyl 2 isotype rat IgG2b CD45R B220 isotype rat IgG2a and CD49b DX5 isotype rat IgM 2 mL Anti Biotin MicroBeads MicroBeads conjugated to monoclonal anti biotin antibody clone Bio3 18E7 2 isotype mouse IgG1 2 mL CD8a Ly 2 MicroBeads MicroBeads conjugated to monoclonal anti mouse CD8a antibody Ly 2 isotype rat IgG2a Capacity For 2x10 total cells up to 100 separations Product format Allcomponentsare supplied in buffer containing stabilizer and 0 05 sodium azide Store protected from light at 2 8 C Do not freeze The expiration date is indicated on the vial label Storage Miltenyi Biotec Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 CD82. Dendritic Cell Isolation Kit mouse Order no 130 091 169 1 1 Principle of the MACS Separation The isolation of CD8 mouse dendritic cells is performed in a two step procedure First T B and NK cells are indirectly magnetically 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 CD8 dendritic cells are directly labeled with CD8a Ly 2 MicroBeads and isolated by positive selection from the pre enriched dendritic cell fraction The unlabeled cells run through this cell fraction is thus depleted of CD8 cells After removing the column from the magnetic field the magnetically retained CD8 cells can be eluted as the positively selected cell fraction To increase the purity the positively selected cell fraction containing the CD8 cells is separated over a second column Single cell suspension Depletion of T B and NK cells 1 Indirect magnetic labeling of T B and NK cells with Biotin Antibody Cocktail and Anti Biotin MicroBeads 2 Magnetic separation using LD Column autoMACs or autoMACS Pro program Depl025 Flow through fraction pre enriched dendritic cells Positive selection of CD8 dendritic cells 1 Direct magnetic labeling of CD8 dendritic cells with CD8a Ly 2 MicroBeads 2 Magnetic separation using two MS Columns autoM ACS or autoM ACS Pro program Possel
3. CD11c dendritic cells CD11c FITC CD8 PE Isolated CD8 CD11c dendritic cells CD11c FITC CD8 PE References gt 1 Shortman K and Liu YJ 2002 Mouse and human dendritic cell subtypes Nat Rev Immunol 2 3 151 161 2 Anjuere F et al 1999 Definition of dendritic cell subpopulations present in the spleen Peyer s patches lymph nodes and skin of the mouse Blood 93 2 590 598 3 Vremec D et al 2000 CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen J Immunol 164 2978 2986 4 Henri S et al 2001 The dendritic cell populations of mouse lymph nodes J Immunol 167 741 748 All protocols and data sheets are available at www miltenyibiotec com 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 Mil
4. d2 Elution from column CD8 dendritic cells 1 2 Background information Dendritic cells are a rare heterogeneous population of hematopoietic cells This kit was designed for the easy isolation of CD8 dendritic cells which constitute one of the three major dendritic cell subpopulations in mouse spleen CD8 dendritic cells express CD11c CD205 MHC class II CD40 CD80 and CD86 and are negative for CD4 and CD11b In spleen and lymph node CD8 dendritic cells are found at moderate levels 23 and 17 of all CD11c dendritic cells respectively and are located in the T cell areas Skin draining lymph nodes harbor an additional CD8 dendritic cell subset CD4 CD8t tCD11b CD205 which accounts for 33 of all CD11c dendritic cells in this organ and is thought to represent the mature form of Langerhans cells www miltenyibiotec com A Miltenyi Biotec Inc 12740 Earhart Avenue Auburn CA 95602 USA Phone 800 FOR MACS 1 530 888 8871 Fax 1 530 888 8925 page 1 4 0 788 000 0F L 1 3 Applications antigen uptake and antigen processing T cell activation or T cell tolerance induction cross priming of cytotoxic T cells T helper cell polarization by CD8 dendritic cells 4 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 Ri
5. e Order no 130 091 169 O 2 4 Magnetic labeling of CD8 dendritic cells A Volumes for magnetic labeling given below are for an initial starting cell number of 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 1 Centrifuge cell suspension at 300xg for 10 minutes Aspirate supernatant completely 2 Resuspend cell pellet in 400 uL of buffer per 10 total cells 3 Add 100 uL of CD8a Ly 2 MicroBeads per 10 total cells 4 Mix well and incubate for 30 minutes on ice A Note Mix cells once during the incubation time 5 Wash cells by adding 5 10 mL of buffer per 10 cells and centrifuge at 300xg for 10 minutes at 2 8 C Aspirate supernatant completely 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 CD8 dendritic cells mm Positive selection with MS Columns A To achieve highest purities always perform two cosecutive column runs 1 Place column in the magnetic field of a suitable MACS Separator For details see the respective MACS Column data sheet 2 Prepare column by rinsing with 500 uL of buffer 3 Apply cell suspension onto the column 4 Collect unlabeled cells that pass through and wash column with 3x500 uL of buffer Collect total effluent this is the unlabel
6. e and provide tubes for collecting the labeled and unlabeled cell fractions Place sample tube at the uptake port and the fraction collection tubes at port negl and port pos2 3 Fora standard separation choose the following program Positive selection Posseld2 Collect positive fraction from outlet port pos2 Magnetic separation with the autoMACS Pro Separator 1 Prepare and prime the instrument 2 Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions Place sample tube in row A of the tube rack and the fraction collection tubes in rows B and C 3 Fora standard separation choose the following program Positive selection Posseld2 Collect positive fraction in row C of the tube rack 3 Example of a separation using the CD8 Dendritic Cell Isolation Kit CD8 dendritic cells were isolated from a mouse spleen using the CD8 Dendritic Cell Isolation Kit an LD and two MS Columns a MidiMACS and a MiniMACS Separator Cells are fluorescently stained with CD11c FITC 130 091 842 and CD8a PE 130 091 603 Cell debris and dead cells are excluded from the analysis based on scatter signals and PI fluorescence Before separation CD11c FITC CD8 PE Miltenyi Biotec Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use Order no 130 091 169 Pre enriched
7. ed cell fraction 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 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 6 Pipette 1 mL of buffer onto the column Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column 7 To increase the purity of CD8 cells the eluted fraction must be enriched over a second MS Column Repeat the magnetic separation procedure as described in steps 1 to 6 by using a new column www miltenyibiotec com A page 3 4 0 788 000 0F L Positive selection with the autoMACS Separator or the autoM ACS Pro Separator A Refer to the respective user manual for instructions on how to use the autoMACS Separator or the autoMACS Pro Separator A Buffers used for operating the autoMACS Separator or the autoMACS Pro Separator should have a temperature of 10 C A Program choice depends on the isolation strategy the strength of magnetic labeling and the frequency of magnetically labeled cells For details refer to the section describing the cell separation programs in the respective user manual Magnetic separation with the autoMACS Separator 1 Prepare and prime the instrument 2 Apply tube containing the sampl
8. for flow cytometric analysis e g CD8a FITC 130 091 605 CD8a PE 130 091 603 CD8a APC 1330 091 606 CD11c FITC 130 091 842 CD11c PE 130 091 830 CD1lc APC 130 091 844 For more information about other fluorochrome conjugates see www miltenyibiotec com Optional Propidium iodide PI or 7 AAD for flow cytometric exclusion of dead cells Optional Dead Cell Removal Kit 130 090 101 for the depletion of dead cells Optional Pre Separation Filters 130 041 407 to remove cell clumps Miltenyi Biotec Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use Order no 130 091 169 2 Protocol 2 1 Sample preparation To obtain high numbers of CD8 dendritic cells with high purities from murine spleen or lymph nodes single cell suspensions need to be prepared by enzymatic disaggregation with Collagenase D Protocols which entirely rely on mechanical disruption are not recommended 1 Place isolated spleen in a 6 cm petri dish with sufficient Collagenase D solution to completely cover the bottom of the dish 5 mL spleen 2 Inject mouse spleen with 500 uL of Collagenase D solution per spleen using a 1 mL syringe and a 25G needle then cut the tissue in smaller pieces by using a pair of scissors 3 Incubate the spleen pieces in Collagenase D solution for 30 minutes at 37 C 4 Pass the whole material
9. i e remaining fragments and Collagenase D released cells through a 70 um cell strainer using a plunger 5 Collect all cells in a 50 mL tube and wash the cells by adding buffer to obtain a final volume of 15 mL 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 6 Proceed to magnetic labeling 2 2 O 2 2 Magnetic labeling of T B and NK cells 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 Wet filter with buffer before use 1l Determine cell number 2 Centrifuge cell suspension at 300xg for 10 minutes Aspirate supernatant completely 3 Resuspend cell pellet in 200 uL of buffer
10. ions on how to use the autoMACS Separator or the autoMACS Pro Separator A Buffers used for operating the autoMACS Separator or the autoMACS Pro Separator should have a temperature of 10 C A Program choice depends on the isolation strategy the strength of magnetic labeling and the frequency of magnetically labeled cells For details refer to the section describing the cell separation programs in the respective user manual Magnetic separation with the autoMACS Separator 1 Prepare and prime the instrument 2 Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions Place sample tube at the uptake port and the fraction collection tubes at port negl 3 Fora standard separation choose the following program Depletion Depl025 Collect negative fraction from outlet port negl Magnetic separation with the autoMACS Pro Separator 1 Prepare and prime the instrument 2 Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions Place sample tube in row A of the tube rack and the fraction collection tubes in rows B and C 3 Fora standard separation choose the following program Depletion Depl025 Collect negative fraction in row B of the tube rack Miltenyi Biotec Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic us
11. nsing Solution 130 091 222 Keep buffer cold 2 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 mouse serum albumin mouse serum or fetal bovine serum Buffers or media containing Ca or Mg are not recommended for use MACS Columns and MACS Separators Depletion of T B and NK cells is performed on an LD Column The subsequent positive selection of CD8 dendritic cells is performed on two MS Columns Depletion and positive selection can also be performed by using the autoMACS or the autoMACS Pro Separator Column Max number Max number Separator of labeled cells of total cells Depletion LD 10 5x10 MidiMACS QuadroMACS VarioMACS SuperMACS Positive selection MS 10 2x10 MiniMACS OctoMACS VarioMACS SuperMACS Depletion or positive selection autoMACS 2x 10 4x10 autoM ACS autoMACS Pro 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 Collagenase D 1 mg mL Collagenase D gt 0 15 U mg e g from Roche Diagnostics Germany in 10 mM Hepes NaOH pH 7 4 150 mM NaCl 5 mM KCI 1 mM MgCl 1 8 mM CaCl Optional Fluorochrome conjugated CD8a and CD1ic antibody
12. per 10 total cells 4 Add 50 uL of Biotin Antibody Cocktail per 10 total cells 5 Mix well and incubate for 10 minutes on ice 6 Add 150 uL of buffer and 100 uL of Anti Biotin MicroBeads per 10 total cells 7 Mix well and incubate for 15 minutes on ice www miltenyibiotec com A page 2 4 XO XXX XXX XXX 8 Wash cells by adding 5 10 mL of buffer per 10 cells and centrifuge at 300xg for 10 minutes at 4 C Aspirate supernatant completely 9 Resuspend cell pellet in buffer Depletion with LD Column 500 uL for up to 1 25x10 cells Depletion with autoMACS 500 uL for up to 1x10 cells A Note For larger cell numbers scale up buffer volume accordingly 10 Proceed to magnetic separation 2 3 me 2 3 Magnetic separation Depletion of T B and NK cells 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 that pass through and wash column with 2xlmL of buffer Collect total effluent this is the unlabeled cell fraction Perform washing steps by adding buffer two times Only add new buffer when the column reservoir is empty 5 Proceed to 2 4 for the isolation of CD8 dendritic cells Depletion with the autoMACS Separator or the autoMACS Pro Separator A Refer to the respective user manual for instruct
13. tenyi 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 isa registered trademark and autoMACS MidiMACS MiniMACS OctoMACS QuadroMACS SuperMACS and VarioMACS are trademarks of Miltenyi Biotec GmbH 2008 Miltenyi Biotec GmbH www miltenyibiotec com page 4 4
Download Pdf Manuals
Related Search