CD61 MicroBeads - Miltenyi Biotec
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1. 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 1 Determine cell number 2 Centrifuge cell suspension at 300xg for 10 minutes Aspirate supernatant completely 3 Resuspend cell pellet in 80 uL of buffer per 10 total cells 4 Add 20 uL of CD61 MicroBeads per 10 total cells 5 Mix well and incubate for 15 minutes at 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 Optional Add staining antibodies e g add 10 uL of CD61 PE 130 081 501 and incubate for 5 minutes at 4 8 C 7 Wash cells by adding 1 2 mL of buffer per 10 cells and centrifuge at 300xg for 10 minutes Aspirate supernatant completely 8 Resuspend up to 108 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 25x108 cells in 500 uL2. of buffer 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 130 051 101 9 Proceed to magnetic separation 2 3 nn 2 3 Magnetic separation A Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD61 cells For details see table in section 1 3 Magnetic separation with MS or LS Columns 1 Place column in the magnetic field of a suitable MACS Separator For details see respective MACS Column data sheet 2 Prepare column by rinsing with appropriate amount of buffer MS 500 uL LS 3 mL 3 Apply cell suspension onto the column 4 Collect unlabeled cells that pass through and wash column with appropriate amount of buffer Perform washing steps by adding buffer three times Only add new buffer when the column reservoir is empty MS 3x500 uL LS 3x3 mL Collect total effluent this is the unlabeled cell fraction 5 Remove column from the separator and place it on a suitable collection tube 6 Pipette an 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 A Note To increase the purity of the magnetically labeled fraction it can be passed over a new freshly prepared column Magnetic separation with XS Columns For instruc
3. 30 091 222 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 human serum albumin human serum or fetal calf serum Buffers or media containing Ca or Mg are not recommended for use MACS Columns and MACS Separators CD61 cells can be enriched from bone marrow peripheral blood or cultivated cells by using MS LS or XS Columns positive selection CD61 MicroBeads can be used for depletion of CD61 cells on LD CS or D Columns Cells which strongly express the CD61 antigen can also be depleted using MS LS or XS Columns Positive selection or depletion can also be performed by using the autoMACS Separator Column Max number Max number Separator of labeled cells of total cells Positive selection MS 10 2x108 MiniMACS OctoMACS VarioMACS SuperMACS LS 10 2x10 MidiMACS QuadroMACS VarioMACS SuperMACS XS 107 2x10 SuperMACS Depletion LD 108 5x108 MidiMACS QuadroMACS VarioMACS SuperMACS CS 2x108 VarioMACS SuperMACS D 10 SuperMACS Positive selection or depletion 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 www m
4. 90 L9 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 Sample preparation 2 2 Magnetic labeling 2 3 Magnetic separation Example of a separation using CD61 MicroBeads 4 References 5 Special cell separation protocol Megakaryocyte isolation 1 Description Components 2 mL CD61 MicroBeads human MicroBeads conjugated to monoclonal mouse anti human CD61 antibodies isotype mouse IgGl Size For 10 total cells up to 100 separations Product format CD61 MicroBeads are supplied as a suspension 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 1 1 Principle of MACS Separation First the CD61 cells are magnetically labeled with CD61 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 CD61 cells are retained on the column The unlabeled cells run through and this cell fraction is depleted of CD61 cells After removal of the column from the magnetic field the magnetically retained CD61 cells can be eluted as the positively selected cell fraction 1 2 Background and product applications CD61 MicroBeads can be used for the isolation or depletion of megakaryocytes and their precursors fro
5. ation choose one of the following separation programs Positive selection Possel Depletion Depletes A Note Program choice depends on the isolation strategy the strength of magnetic labeling and the frequency of magnetically labeled cells For details see autoMACS User Manual autoMACS Cell Separation Programs 3 When using the program Possel collect positive fraction outlet port pos1 This is the purified CD61 cell fraction When using the program Depletes collect unlabeled fraction from outlet port neg1 This is the CD61 cell fraction 3 Example of a separation using CD61 MicroBeads Separation of PBMCs using CD61 MicroBeads and a MiniMACS Separator with an MS Column The cells are fluorescently stained with CD61 PE 130 081 501 Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence ma ma aa a ah m limn aam m m m an aa a CD61 platelets CD61 PBMCs Relative cell number CD61 PE PBMCs before separation 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 130 051 101 4 References l Schmitz B et al 1994 Eur J Hematol 52 267 275 104 2 Komor M et al 2005 Stem cells 23 1154 1169 3 Appel S et al 2006 Blood 107 3265 3270 Warnings Reagents contain sodium azide Under acidic condit
6. iltenyibiotec com A Miltenyi Biotec Inc 12740 Earhart Avenue Auburn CA 95602 USA Phone 800 FOR MACS 530 888 8871 Fax 530 888 8925 page 1 4 90 L9 000 0rL Optional Fluorochrome conjugated CD61 antibody for flow cytometric analysis e g CD61 PE 130 081 501 CD45 FITC 130 080 202 CD45 PE 130 080 201 CD45 APC 130 091 230 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 When working with anticoagulated peripheral blood or buffy coat peripheral blood mononuclear cells PBMCs should be isolated by density gradient centrifugation e g using Ficoll Paque For details see section General Protocols in the User Manuals or visit www miltenyibiotec com protocols When working with tissues prepare a single cell suspension by a standard preparation method For details see section General Protocols in the User Manuals or visit www miltenyibiotec com protocols A Note 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 O 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
7. ions 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 MACS is a registered trademark of Miltenyi Biotec GmbH autoMACS MidiMACS MiniMACS OctoMACS QuadroMACS SuperMACS and VarioMACS are trademarks of Miltenyi Biotec GmbH Ficoll Paque and Percoll are trademarks of GE Healthcare companies 2006 Miltenyi Biotec GmbH Printed in Germany www miltenyibiotec com page 3 4 90 L9 000 0rL 5 Special cell separation protocol Megakaryocyte isolation Megakaryocytes can be isolated from bone marrow preparations using CD61 MicroBeads which are developed for separation of human cells based on the expressi
8. m bone marrow and for the removal of platelets from peripheral blood cell preparations The CD61 antigen is also known as the integrin 63 subunit CD61 combines with CD41 to form the heterodimeric gplIIb gpHIa complex which is present on human megakaryocytes and platelets mediating cell adhesion processes Together with CD51 CD61 forms the vitronectin receptor which is present on platelets as well as on a variety of other cell types like osteoclasts and endothelial vessel cells CD61 MicroBeads were reported to cross react with canine platelets Miltenyi Biotec Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 CD61 MicroBeads human Order no 130 051 101 Examples of applications CD61 cells from bone marrow cells are isolated for studies on megakaryocytopoiesis Positive selection or depletion of cells expressing human CD61 antigen Isolation of megakaryocytes from human bone marrow see 5 Special cell separation protocol Megakaryocytes isolation Depletion or isolation of platelets from human peripheral blood Isolation or depletion of leukocyte platelet conjugates from human peripheral blood 1 3 Reagent and instrument requirements Buffer Prepare a solution containing phosphate buffered saline PBS pH 7 2 0 5 BSA and 2 mM EDTA by diluting MACS BSA Stock Solution 130 091 376 1 20 in autoMACS Rinsing Solution 1
9. n spongiosa material e g from ribs 2 Take up spongiosa material in 5 mL of RPMI 1640 per approximately 0 5 cm of spongiosa material 3 Incubate cells in 6 well plate at 37 C 5 CO2 for 4 hours under agitation to detach the bone marrow cells from the bone 4 Subsequently incubate cells overnight in 25 cm tissue flask at 37 C 5 CO to eliminate stroma cells and fibroblasts Collect the non adherent cells 5 Wash cells by adding RPMI FCS EDTA centrifuge at 200xg for 10 minutes remove supernatant completely Repeat washing procedure once and resuspend cells in 7 mL RPMI FCS EDTA 6 Carefully layer 7 mL of diluted cell suspension over 7 mL of Percoll 1 05 g mL 7 Centrifuge for 30 minutes at 400xg at 20 C in a swinging bucket rotor without break 8 Aspirate the upper layer leaving the mononuclear cell layer undisturbed at the interphase 9 Carefully collect interphase cells and wash them in PBS BSA EDTA Centrifuge for 10 minutes at 200xg at 20 C Repeat this wash step 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 130 051 101 Magnetic labeling of 1x107 total cells in suspension O 1 Resuspend 1x10 cells with PBS BSA EDTA in a final volume of 80 uL 2 Add 20 uL CD61 MicroBeads 3 Mix well and incubate for 15 minutes at 4 8 C 3 Optional Add fl
10. on of the CD61 antigen CD61 is expressed on thrombocytes megakaryocytes osteoclasts and endothelial vessel cells For MACS separation megakaryocytes are magnetically labeled using CD61 MicroBeads The magnetically labeled cells are retained on a MACS Column in the magnetic field of a MACS Separator Instrument and reagent requirements Buffer Prepare a solution containing phosphate buffered saline PBS pH 7 2 0 5 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 human serum albumin human serum or fetal calf serum Buffers or media containing Ca or Mg are not recommended for use CD61 MicroBeads 130 051 101 Large Cell Separation Columns 130 042 202 RPMI 1640 130 091 440 RPMI FCS EDTA RPMI 1640 containing 25 FCS and 2 mM EDTA Percoll 1 05 g mL Optional Fluorochrome conjugated CD61 antibody for flow cytometric analysis e g CD61 PE 130 081 501 Isolation of megakaryocytes from bone marrow A If cells cannot be separated on the day of harvest store cells at 4 C A Remove all cell clumps during the cell preparation 1 Collect huma
11. tions 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 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 Collect total effluent This is the unlabeled cell fraction Depletion with CS Columns 1 Assemble CS Column and place it in the magnetic field of a suitable MACS Separator For details see 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 see CS Column data sheet 3 Apply cell suspension onto the column 4 Collect unlabeled cells which pass through and wash column with 30 mL buffer from the top Collect total effluent This is the unlabeled cell fraction www miltenyibiotec com A page 2 4 90 L9 000 0rL Depletion with D Columns For instructions on column assembly and separation refer to the D Column data sheet Magnetic separation with the autoMACS Separator A Refer to the auttoMACS User Manual for instructions on how to use the autoMACS Separator 1 Prepare and prime autoMACS Separator 2 Place tube containing the magnetically labeled cells in the autoMACS Separator For a standard separ
12. uorochrome conjugated CD61 antibody e g CD61 PE 130 081 501 at the titer recommended by the manufacturer and incubate for an additional 5 10 minutes at 4 8 C 4 Wash cells by adding 1 2 mL of buffer per 10 cells and centrifuge at 300xg for 10 minutes Aspirate supernatant completely 5 Resuspend up to 108 cells in 500 uL of buffer nm Magnetic separation 1 Place Large Cell Separation Column without flow resistor in the MACS Separator 2 Prepare column by rinsing with 500 uL of PBS BSA EDTA 3 Apply cell suspension onto the column 4 Collect unlabeled cells which pass through and wash column with 3x500 uL of PBS BSA EDTA Perform washing steps by adding buffer three times each time once the column reservoir is empty Collect total effluent This is the unlabeled cell fraction 5 Remove column from the separator and place it on a suitable collection tube 6 Pipette appropriate 1 mL of PBS BSA EDTA onto the column Immediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column A Note To increase the purity of the magnetically labeled fraction it can be passed over a new freshly prepared column 7 Elute positive fraction as described above and proceed to analysis by flow cytometry or fluorescence microscopy www miltenyibiotec com A page 4 4
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