Anti-Biotin 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 bovine serum FBS Buffers or media containing Ca or Mg are not recommended for use MACS Columns and MACS Separators Labeled material can be enriched by using MS LS or XS Columns or depleted with the use of LD CS or D Columns If labeling with biotinylated antibodies or molecules is sufficiently strong magnetically labeled biological material can efficiently be depleted by using MS LS or XS Columns Positive selection or depletion can also be performed by using the autoMACS Pro or the autoMACS Separator Column Max number Max number Separator of labeled cells of total cells Positive selection MS 10 2x10 MiniMACS OctoMACS VarioMACS SuperMACS II LS 10 2x10 MidiMACS QuadroMACS VarioMACS SuperMACS II XS 10 2x10 SuperMACS II Depletion LD 10 5x10 MidiMACS QuadroMACs VarioMACS SuperMACS II cs 2x10 VarioMACS SuperMACS II D 10 SuperMACS II Positive selection or depletion autoMACS 2x10 4x10 autoMACS Pro autoMACS A Note Column adapters are required to insert certain columns into the VarioMACS or SuperMACS II Separators For details refer to the respective MACS Separator data sheet Biotin conjugated antibody peptide or ligand Miltenyi2. 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 4 60 7 S 000 0F L Optional FcR Blocking Reagent human 130 059 901 mouse 130 092 575 to avoid Fc receptor mediated antibody labeling when using human or mouse samples Optional Fluorochrome conjugated anti biotin antibody for flow cytometric analysis e g Anti Biotin FITC 130 090 857 Anti Biotin PE 130 090 756 or Anti Biotin APC 130 090 856 For more information about fluorochrome conjugates refer to www miltenyibiotec com Optional Propidium Iodide Solution 130 093 233 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 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 for example using Ficoll Paque 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 aspi
3. 04 The Cell Surface Localized Heat Shock Protein 70 Epitope TKD Induces Migration and Cytolytic Activity Selectively in Human NK Cells J Immunol 172 972 980 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 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 2010 Miltenyi Biotec GmbH All rights reserved U
4. 607 S 000 0F L Miltenyi Biotec 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 2 3 Magnetic separation 3 Example of a separation using the Anti Biotin MicroBeads 4 References 1 Description Components 2 mL Anti Biotin MicroBeads MicroBeads conjugated to monoclonal mouse anti biotin antibodies isotype mouse IgG1 Capacity For 10 total cells up to 100 separations Product format Anti Biotin MicroBeads are supplied in buffer containing stabilizer and 0 05 sodium azide Storage 1 1 Principle of the MACS Separation First the biological material of interest e g cells bacteria or subcellular material is labeled with biotinylated antibodies or ligands Subsequently the material is magnetically labeled with Anti Biotin 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 material is retained within the column The unlabeled material runs through this cell fraction is thus depleted of magnetically labeled material After removing the column from the magnetic field the magnetically retained material can be eluted as the positively selected cell fraction To increase the purity the positively selected cell fraction containing the magnetically labeled c
5. d place it on a suitable collection tube 6 Pipette the 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 mi 7 Optional To increase the purity of magnetically labeled cells the eluted fraction can be enriched over a second MS or LS Column Repeat the magnetic separation procedure as described in steps 1 to 6 by using a new 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 refer to the 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 Order no 130 090 485 Depletion with CS Columns 1 Assemble CS Column and place it in the magnetic field of a suitable MACS Separator For details refer to the 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 refer to the CS Column data sheet 3 Apply ce
6. ed cell fractions Place sample tube at the uptake port and the fraction collection tubes at port negl and the appropriate port for the positive fraction 3 Choose an appropriate program according to the recommendations in the autoMACS Separator user manual Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 3 4 60 7 S 000 0F L 3 Example of a separation using the Anti Biotin MicroBeads Separation of human PBMCs using biotinylated mouse anti human CD3 antibody the Anti Biotin MicroBeads an MS Column and a MiniMACS Separator Cells are fluorescently stained with Anti Biotin PE 130 090 756 Cell debris and dead cells are excluded from the analysis based on scatter signals and propidium iodide fluorescence PBMCs before separation CD3 Biotin Anti Biotin PE Forward scatter Negative fraction depleted of CD3 cells CD3 Biotin Anti Biotin PE Forward scatter Positive fraction of enriched CD3 cells CD3 Biotin Anti Biotin PE Forward scatter Order no 130 090 485 4 References 1 Lehmann J et al 2002 Expression of the integrin aEb7 identifies unique subsets of CD25 as well as CD25 regulatory T cells Proc Natl Acad Sci USA 99 13031 13036 2 Lang R et al 2003 SOCS3 regulates the plasticity of gp130 signaling Nature Immunol 4 546 550 3 Gastpar R et al 20
7. ells can be separated over a second column 1 2 Background information Anti Biotin MicroBeads have been developed for indirect magnetic labeling and separation of cells or other biological materials that are labeled with a biotinylated primary antibody or ligand or with a cocktail of biotinylated antibodies The biotinylated molecule is recognized by a monoclonal anti biotin antibody coupled to MicroBeads Anti Biotin MicroBeads have the advantage of not binding to free biotin which is often present in culture media This feature provides the most sensitive labeling and separation of cells Store protected from light at 2 8 C Do not freeze The expiration date is indicated on the vial label Anti Biotin MicroBeads Order no 130 090 485 1 3 Applications Positive selection or depletion also of cells with low antigen expression Some examples for the use of Anti Biotin MicroBeads are the separation of murine ag CD25 and ag CD25 T cell subsets isolation of CD45 2 cells from the bone marrow of chimeric mice or the enrichment of human CD94 NK cells 1 4 Reagent and instrument requirements e Buffer Prepare a solution containing phosphate buffered saline PBS pH 7 2 0 5 bovine serum albumin BSA and 2mM EDTA by diluting MACS BSA Stock Solution 130 091 376 1 20 with autoMACS Rinsing Solution 130 091 222 Keep buffer cold 2 8 C Degas buffer before use as air bubbles could block the column
8. es and or longer incubation times may lead to non specific cell labeling Working on ice may require increased incubation times 10 11 12 Order no 130 090 485 Determine cell number Optional Add FcR Blocking Reagent human or mouse in appropriate ratio For details refer to the respective FcR Blocking Reagent data sheet Label cells with the biotinylated antibody using time and titer according to manufacturer s instructions Typically staining for 5 minutes is sufficient A Note The biotinylated antibody should be used at its optimal titer i e with optimal staining intensity and no background staining Wash cells by adding 1 2 mL of buffer per 10 cells and centrifuge at 300xg for 10 minutes Aspirate supernatant completely Repeat step 4 Resuspend cell pellet in appropriate amount of buffer per 10 total cells When labeling without using FcR Blocking Reagent resuspend in 80 uL of buffer When MACS FcR Blocking Reagent human has been used resuspend in 60 uL of buffer When MACS FcR Blocking Reagent mouse has been used resuspend in 70 uL of buffer Add 20 uL of Anti Biotin MicroBeads per 10 total cells Mix well and incubate for 15 minutes in the refrigerator 2 8 C Optional Add staining antibodies e g 10 uL ofAnti Biotin FITC 130 090 857 and incubate for 5 minutes in the dark in the refrigerator 2 8 C Wash cells by adding 1 2 mL of buffer per 10 cells and centrif
9. ll suspension onto the column 4 Collect unlabeled cells that 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 Magnetic separation with the autoMACS Pro Separator or the autoM ACS Separator A Refer to the respective user manual for instructions on how to use the autoM ACS Pro Separator or the autoMACS Separator A Buffers used for operating the autoMACS Pro Separator or the autoMACS Separator should have a temperature of 210 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 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 Choose an appropriate program according to the recommendations in the autoMACS Pro Separator 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 unlabel
10. nless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 4 4
11. rate supernatant Repeat washing step When working with tissues or lysed blood prepare a single cell suspension using standard methods When working with mouse lymphoid organs mouse non lymphoid tissues or mouse peripheral blood prepare a single cell suspension using manual methods or the gentleMACS Dissociator For details refer to the protocols section at www miltenyibiotec com protocols A 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 a 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 labeling Pass cells through 30 um nylon mesh Pre Separation Filters 130 041 407 to remove cell clumps which may clog the column Moisten filter with buffer before use A The recommended incubation temperature is 2 8 C Higher temperatur
12. uge at 300xg for 10 minutes Aspirate supernatant completely Resuspend up to 10 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 25x10 cells in 500 uL of buffer Proceed to magnetic separation 2 3 Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use page 2 4 607 S 000 0F L nm 2 3 Magnetic separation A Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of magnetically labeled cells For details refer to the table in section 1 4 A Always wait until the column reservoir is empty before proceeding to the next step 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 the appropriate amount of buffer MS 500 uL LS 3 mL 3 Apply cell suspension onto the column Collect flow through containing unlabeled cells 4 Wash column with the appropriate amount of buffer Collect unlabeled cells that pass through and combine with the effluent from step 3 MS 3x500 uL LS 3x3 mL A Note Perform washing steps by adding buffer aliquots only when the column reservoir is empty 5 Remove column from the separator an
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