CD34 MicroBead Kit
Contents
1. for research use only and not for diagnostic or therapeutic use page 3 4 S0 0S6 L00 0rL 3 Example of a separation using the CD34 MicroBead Kit Isolation of CD34 cells from PBMCs using the CD34 MicroBead Kit two MS Columns and a MiniMACS Separator Cells were stained with CD34 PE 130 081 002 and CD45 FITC 130 080 202 Cell debris and dead cells are excluded from the analysis based on scatter signals and propidium iodide fluorescence Before separation CD45 FITC CD34 PE CD34 cells CD45 FITC CD34 PE 4 References 1 Giarratana M C et al 2005 Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells Nat Biotechnol 23 69 74 2 Timmermans F et al 2007 Endothelial outgrowth cells are not derived from CD133 cells or CD45 hematopoietic precursors Arterioscler Thromb Basc Biol 27 1572 1579 3 O E et al 2011 Efficient nonadhesive ex vivo expansion of early endothelial progenitor cells derived from CD34 human cord blood fraction for effective therapeutic vascularization FASEB J 25 159 169 4 Wang Z Z et al 2007 Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo Nat Biotechnol 25 317 318 5 Bonanno G et al 2009 Interleukin 21 induces the differentiation of human umbilical cord blood CD34 lineage cells into pseudomature lytic NK cells BMC Immunol 10 46 All protocols and data s2. CS SuperMACS II XS 10 2x10 SuperMACS II Positive selection 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 Optional MC CD34 Stem Cell Cocktail 130 093 427 for flow cytometric analysis of separated cells Optional Fluorochrome conjugated antibodies for flow cytometric analysis e g CD34 FITC 130 081 001 CD34 PE 130 081 002 CD34 APC 130 090 954 CD133 293C3 PE 130 090 853 CD133 293C3 PE 130 090 854 CD45 FITC 130 080 202 CD45 PE 130 080 201 or CD45 APC 130 091 230 For more information about antibodies refer to www miltenyibiotec com antibodies 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 30 um 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 Carefu
3. S0 0S6 L00 0rL Miltenyi Biotec Contents 1 Description 1 1 Principle of the MACS Separation 1 2 Background information 1 3 Applications 1 4 Reagent and instrument requirements 2 Protocol 2 1 Sample preparation 2 2 Magnetic labeling 2 3 Magnetic separation 2 4 Optional Evaluation of hematopoietic progenitor cell purity Example of a separation using the CD34 MicroBead Kit 4 References 1 Description 2 mL CD34 MicroBeads human MicroBeads conjugated to monoclonal mouse anti human CD34 antibodies isotype mouse IgGl 2 mL FcR Blocking Reagent human Human IgG Components or 10 mL CD34 MicroBeads human MicroBeads conjugated to monoclonal mouse anti human CD34 antibodies isotype mouse IgGl 10 mL FcR Blocking Reagent human Human IgG Capacity or 10 mL For 10 total cells up to 100 separations Product format CD34 MicroBeads are 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 GmbH Friedrich Ebert StraRe 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 macs miltenyibiotec de www miltenyibiotec com 2 mL For 2x10 total cells up to 20 separations CD34 MicroBead Kit human 2 mL 10 mL 130 046 702 130 046 703 1 1 Principle of the MACS Separation First the CD34 cells are m
4. agnetically labeled with CD34 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 CD34 cells are retained within the column The unlabeled cells run through this cell fraction is thus depleted of CD34 cells After removing the column from the magnetic field the magnetically retained CD34 cells can be eluted as the positively selected cell fraction 1 2 Background information The CD34 antigen is a single chain transmembrane glycoprotein expressed on human hematopoietic progenitor cells endothelial progenitor cells vascular endothelial cells embryonic fibroblasts and some cells in fetal and adult nervous tissue The CD34 MicroBead Kit contains MicroBeads directly conjugated to CD34 antibodies for magnetic labeling of CD34 expressing cells from peripheral blood cord blood bone marrow apheresis harvest or differentiated ES and iPS cells Hematopoietic progenitor cells present at a frequency of about 0 05 0 2 in peripheral blood 0 1 0 5 in cord blood and 0 5 3 in bone marrow can be rapidly and efficiently enriched 1 3 Applications Positive selection or depletion of cells expressing human CD34 antigen Isolation of hematopoietic progenitor cells Isolation of endothelial progenitor cells EPCs Isolation of CD34 progenitor cells from differentiated ES and iPS cell cultures Invitro differentia
5. heets are available at www miltenyibiotec com Order no 130 046 702 Order no 130 046 703 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 2013 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
6. lly aspirate supernatant Repeat washing step When working with tissues or lysed blood prepare a single cell suspension using standard methods 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 Order no 130 046 702 Order no 130 046 703 Preparation of cells from leukapheresis material 1 Filter apheresis harvest through 30um nylon mesh Pre Separation Filters 30 um 130 041 407 in order to remove cell clumps 2 Wash cells once with buffer and resuspend in a final volume of 300 uL of buffer for up to 10 cells Proceed to magnetic labeling e 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 Separa
7. nstrument 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 and port pos2 3 For a standard separation choose one of the following programs Positive selection of CD34 cells from peripheral blood bone marrow or leukapheresis Posseld Positive selection of CD34 cells from cord blood Posseld2 Collect positive fraction from outlet port pos2 2 4 Optional Evaluation of hematopoietic progenitor cell purity The purity of the isolated hematopoietic progenitor cells can be evaluated by flow cytometry or fluorescence microscopy Analysis of CD34 cells can be accomplished by direct immunofluorescent staining using an antibody recognizing an epitope different from that recognized by the CD34 monoclonal antibody QBEND 10 e g CD34 PE clone AC136 130 081 002 For optimal discrimination of CD34 cells from other leukocytes counterstain cells with an antibody against CD45 e g CD45 FITC 130 080 202 CD34 cells express CD45 at a lower level as compared to lymphocytes Use the antibodies in appropriate concentrations as recommended by the manufacturers Typically staining for 5 minutes at 2 8 C should be sufficient After fluorescent staining cells should be washed and resuspended in buffer Unless otherwise specifically indicated Miltenyi Biotec products and services are
8. s 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 Magnetic separation with the autoMACS Pro Separator or the autoMACS 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 autoM ACS 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 Order no 130 046 702 Order no 130 046 703 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 For a standard separation choose one of the following programs Positive selection of CD34 cells from peripheral blood bone marrow or leukapheresis Posseld Positive selection of CD34 cells from cord blood Posseld2 Collect positive fraction in row C of the tube rack Magnetic separation with the autoMACS Separator 1 Prepare and prime the i
9. tion Filters 30 um 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 temperatures and or longer incubation times may lead to non specific cell labeling Working on ice may require increased incubation times 1 Determine cell number 2 Centrifuge cell suspension at 300xg for 10 minutes Aspirate supernatant completely 3 Resuspend cell pellet in 300 uL of buffer for up to 10 total cells 4 Add 100 uL of FcR Blocking Reagent for up to 10 total cells 5 Add 100 uL of CD34 MicroBeads for up to 10 total cells 6 Mix well and incubate for 30 minutes in the refrigerator 2 8 C 7 Optional Add fluorochrome conjugated CD34 antibody recognizing another epitope than QBEND 10 e g clone AC136 CD34 PE 130 081 002 or fluorochrome conjugated CD45 antibody e g CD45 FITC 130 080 202 and incubate for 5 minutes in the dark in the refrigerator 2 8 C 8 Wash cells by adding 5 10 mL of buffer for up to 10 cells and centrifuge at 300xg for 10 minutes Aspirate supernatant completely 9 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 10 Proceed to magnetic separation 2 3 Unless otherwise specifically indicated Miltenyi Biotec produc
10. tion studies Studies on hematologic malignancies 1 4 Reagent and instrument requirements Buffer Preparea 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 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 human serum albumin human serum or fetal bovine serum FBS Buffers or media containing Ca or Mg are not recommended for use 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 S0 0S6 L00 0rL MACS Columns and MACS Separators CD34 cells can be enriched by using MS LS or XS Columns positive selection Cells that strongly express the CD34 antigen can also be depleted 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 total cells of labeled cells Positive selection MS 10 2x108 MiniMACS OctoMACS VarioMACS SuperMACS II LS 10 2x10 MidiMACS QuadroMACS VarioMA
11. ts and services are for research use only and not for diagnostic or therapeutic use page 2 4 S0 0S6 L00 0rL aE 2 3 Magnetic separation A Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD34 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 flow through from step 3 MS 3x500 uL LS5S 3x3 mL A Note Perform washing steps by adding buffer aliquots only when the column reservoir is empty 5 Remove column from the separator and 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 mL 7 Optional To increase the purity of CD34 cells the eluted fraction can be enriched over a second MS or LS Column Repeat the magnetic separation procedure a
12. use page 4 4
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