PR22 MHC Streptamer Manual_0005_2012 Version 2
Contents
1. To wash away unbound MHC magnetic beads are bound and washed on a MS column 8 Wash with 2 ml Buffer IS 9 Add 250 ul Buffer IS and elute retained beads outside of the magnetic field into a fresh vial and firmly flush out the beads using the supplied plunger supplied with the column 10 Centrifuge cell suspension 300 g and resuspend in 250 pl magnetic beads MHC solution Incubate 45 minutes on ice 11 Add 1 5 ml Buffer IS centrifuge cell and beads mixture and wash carefully once with 2 ml Buffer IS e This is necessary to eliminate unbound magnetic beads which may trap cells on the column un specifically 12 Resuspend in 2 ml Buffer IS Proceed to magnetic separation 14 Streptamer Manual Antigen specific staining and functional isolation of T cells 4 2 5 Isolation of antigen specific T cells with Streptamer Magnetic Beads protocol for mouse cells When working with cells from spleen or lymph node cells be careful to resuspend cells completely Other organ preparations may require protease digestion and or gradient centrifugation Mouse T cell separation protocol is established for 2 x 10 cells Higher cell numbers require larger amounts of beads and MHC 1 Centrifuge cells twice 10 minutes at 300 g at 4 C and resuspend in 10 ml Buffer IS respectively 2 Pass cells through a nylon filter tube included in Streptamer Solution Set e This is necessary to remove cell clumps which may clog the col2. 1 2 EDTA is added to a final concentration of 20 mM Same volume of PBS or balanced salt solution is added to the EDTA blood Other anticoagulants have been used like heparin citrate acid citrate dextrose citrate phosphate dextrose 4 2 2 Ficoll gradient centrifugation Procedure for isolation of lymphocytes from blood samples Example GE Healthcare Ficoll Paque Plus 6 x 500 ml 17 1440 03 For details please refer to the included protocol Briefly The required volume of Ficoll 3 ml for 4 ml diluted anticoagulated blood sample is aseptically withdrawn using a syringe Ficoll Paque Plus 3 ml is added to a centrifuge tube Carefully layer diluted blood sample 4 ml on Ficoll Paque Plus Important When layering the sample do not mix Ficoll and diluted blood sample Centrifuge at 400x g for 30 40 minutes at 18 20 C Draw off the upper layer using a clean Pasteur pipette leaving the lymphocyte layer undisturbed at the interface Care should be taken not to disturb the lymphocyte layer The upper layer of plasma which is essentially free of cells may be saved for later use Using a clean Pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube It is critical to remove all of the interface but a minimum amount of Ficoll and supernatant Removing excess Ficoll causes granulocyte contamination removing excess supernatant results in platelet and plasma protein contamination Add at least
3. 3 volumes of balanced salt solution to the lymphocytes in the test tube Suspend the lymphocytes by gently drawing them in and out of a the Pasteur pipette Streptamer Manual Antigen specific staining and functional isolation of T cells 9 Centrifuge at 10 100 x g and 18 20 C for 10 minutes 10 Remove the supernatant 11 The lymphocytes should now be suspended in an appropriate medium and can be frozen Streptamer Manual Antigen specific staining and functional isolation of T cells 11 4 2 3 Optional CD8 enrichment CD8 enrichment is recommended for the isolation of mouse antigen specific T cells only A detailed protocol is available with the kit i e Miltenyi 130 091 154 Here a brief version 4 2 3 1 Magnetic labeling 1 Determine cell number 2 Centrifuge cell suspension at 300 x g for 10 minutes Pipette off supernatant completely Resuspend cell pellet in 40 ul of buffer per 10 total cells Add 10 pl of biotin antibody cocktail per 10 total cells Mix well and incubate for 10 minutes at 4 8 C Add 30 ul of buffer per 10 total cells Add 20 pl of anti biotin MicroBeads per 10 total cells Mix well and incubate for an additional 15 minutes at 4 8 C o ON DO HU PF Ww Wash cells with buffer adding 10 20 x labeling volume and centrifuge at 300 x g for 10 minutes Pipette off supernatant completely 10 Resuspend cells in 500 pl buffer Up to 10 total cells can b
4. please refer to the protocol enclosed with the columns Place LS column in the magnetic field and prepare column by rinsing with 3 ml Buffer IS Apply cell suspension see Protocol 4 2 4 step 12 or 4 2 5 step 11 respectively onto the column Allow cells to pass through and collect effluent for later analysis Flow through Wash column with 2 x 3 ml Buffer IS Add 2 x 3 ml Buffer IS and elute retained cells outside of the magnetic field into a fresh vial optional take sample for analysis Rinse MS column with 0 5 ml and apply cells eluted from LS column Wash column with 2 x 2 ml Buffer IS Add 2 x 3 ml Buffer IS and elute retained cells outside of the magnetic field into a fresh vial take sample for analysis eluted fraction should contain the isolated T cells Streptamer Manual Antigen specitic staining and functional isolation of T cells 4 2 7 Magnetic separation with the autoMACS separator For detailed instructions on how to use the autoMACS refer to the corresponding user manual 1 Prepare and prime the autoMACS 2 Place tube containing the magnetically labeled cells see Protocol 4 2 4 step 12 or 4 2 5 step 11 respectively in the autoMACS separator and choose program Posseld 3 Collect positive fraction outlet port pos2 This fraction represents the magnetically labeled antigen specific T cells 4 Optional Collect negative fraction outlet port
5. IS 8 06 mM _ Na HPO 0 5 BSA w v 1 47 mM KH PO in PBS pH 7 4 137 mM NaCl pH 7 4 Biotin stock solution isotonic 100 mM biotin NaCl pH 7 4 18 Streptamer Manual Antigen specific staining and functional isolation of T cells 6 References Knabel M Franz T J Schiemann M Wulf A Villmow B Schmidt B Bernhard H Wagner H and Busch D 2002 Reversible MHC multimer staining for functional isolation of T cell populations and effective adoptive transfer Nature Medicine 8 6 631 637 Neudorter J Schmidt B Huster K M Anderl F Schiemann M Holzapfel G Schmidt T Germeroth L Wagner H Peschel C Busch D and Bernhard H 2007 Reversible HLA multimers Streptamer for the isolation of human cytotoxic T lymphocytes functionally active against tumor and virus derived antigens JIM 320 119 131 Wang X Simeoni L Lindquist J A Saez Rodriguez J Ambach A Gilles E D Kliche S and Schraven B 2008 Dynamics of proximal signaling events after TCR CD8 mediated induction of proliferation or apoptosis in mature CD8 T cells J Immunology 180 6704 6712 Yao J Bechter C Wiesneth M H rter G G tz M Germeroth L Guillaume P Hasan F von Harsdorf S Mertens T Michel D D hner H Bunjes D Schmitt M and Schmitt A 2008 Multimer staining of cytomegalovirus phosphoprotein 65 specific T cells for diagnosis and therape
6. d the temperature before starting the protocol 3 2 1 Titration optional e lf the staining protocol is not suitable for your application a titration of the MHC should be performed Our recommendation for the titration is e Test 0 75 ug Strep Tactin PE with 1 ug 2 ug and 5 ug Streptamer MHC class I respectively e The assay can be conducted in a 96 well round bottom microplate e All incubations are carried out in the dark to protect PE from light Streptamer Manual Antigen specific staining and functional isolation of T cells 5 3 2 2 Protocol for the staining of T cells with Streptamers T Incubate 0 75 ug 5 ul Strep Tactin PE and 1 ug 4 ul Streptamer MHC class in a final volume of 50 ul Buffer IS for 45 minutes Add the pre incubated Strep Tactin PE MHC preparation to the cell pellet Incubate for 45 minutes Wash cells twice with 200 ul Buffer IS Cells are ready for FACS analysis or FACS sorting Important All steps have to be performed at 4 C 3 2 3 Protocol for the subsequent dissociation of Streptamers with D biotin After sorting wash cells twice with 200 ul Butter IS Resuspend cells in 200 ul Buffer IS containing 1 mM D biotin and incubate for 20 minutes Wash cells with 200 ul Buffer IS Incubate for further 20 minutes with Buffer IS containing 1mM D biotin Wash cells 4 times with 200 ul Buffer IS Transfer cells into the appropriate buffer or medium for fur
7. e cells 6 70xx 015 600 ul suff for pur of 1 5 10 human or 7 5 108 mouse cells 6 70xx 050 2 ml suff for pur of 5 10 human or 2 5 10 mouse cells Solution set for Streptamer 6 5600 005 for 5 preps 2 10 cells each Magnetic Beads includes e Buffer IS 6 5600 025 for 25 preps 2 10 cells each e Biotin stock solution 4 1 5 Materials required but not provided Blood or T cell sample Miltenyi columns and separators Centrituge Test tubes 4 1 5 1 For optional staining and FACS analysis EMA Strep Tactin PE and Streptamer MHC class CD8 PE or CD8 FITC antibody CD3 FITC or CD3 APC antibody FACScan 4 1 5 2 For optional Ficoll gradient centrifugation Ficoll PBS or balanced salt solution Pasteur pipettes Syringe with needle Silicone solution Distilled water 4 1 5 3 For optional CD8 enrichment CD8 T cell Isolation Kit II Miltenyi 130 091 154 Streptamer Manual Antigen specific staining and functional isolation of T cells 4 2 Experimental procedure The procedure is optimized to isolate antigen specific T cells from 2x10 peripheral blood mononuclear cells PBMC The first chapters describe the isolation of these cells and depletion of non T cell populations respectively When working with freshly isolated or frozen PBMC proceed to Chapter 4 2 4 for human cells or 4 2 5 for mouse cells respectively 4 2 1 Anticoagulant treatment
8. e resuspended in 500 ul larger numbers require an accordingly larger volume of buffer 11 Proceed to magnetic separation 12 Streptamer Manual Antigen specific staining and functional isolation of T cells 4 2 3 2 Magnetic separation with MS or LS columns 1 Place column in the magnetic field of a suitable MACS separator 2 Prepare column by rinsing with appropriate amount of buffer MS 500 ul LS 3 ml 3 Apply cell suspension onto the column Collect flow through Allow cells to pass through and collect effluent as fraction with unlabeled cells representing the enriched CD8 T cell fraction 4 Wash column with appropriate amount of buffer To wash the column Butter IS added three times when column reservoir is empty MS 500 ul LS 3 ml Collect entire effluent and pool with flow through step 3 This fraction represents the CD8 T cells 5 Optional Elute retained cells outside of the magnetic field This fraction represents the magnetically labeled non CD8 T cells 4 2 3 3 Magnetic separation with the autoMACS separator For detailed instructions on how to use the autoMACS please refer to the corresponding user manual 1 Prepare and prime the autoMACS 2 Place tube containing the magnetically labeled cells in the autoMACS separator and choose program Deplete 3 Collect negative fraction outlet port neg1 This fraction represents the enriched CD8 T cells 4 Optional Collect pos
9. ered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law 2 Streptamer Manual Antigen specific staining and functional isolation of T cells 1 Content Content 3 2 The Streptamer Principle 4 3 Streptamer Fluorescent T cell Labeling and Isolation via FACS 5 3 1 Introduction 5 3 2 Experimental procedure 5 3 2 1 Titration optional 5 3 2 2 Protocol for the staining of T cells with Streptamers 6 3 2 3 Protocol for the subsequent dissociation of Streptamers with D biotin 6 4 Streptamer Magnetic T cell Labeling and Isolation via MACS 7 4 Introduction 7 Purification scheme 7 4 1 2 Recommendations for isolating T cells using Streptamer Magnetic Beads and recombinant Streptamer MHC class 7 4 1 3 Example of an isolation of CMV antigen specific T cells 8 4 1 4 Set of reagents for isolation of antigen specific T cells 9 4 1 5 Materials required but not provided 9 4 2 Experimental procedure 10 4 2 Anticoagulant treatment 10 4 2 2 Ficoll gradient centrifugation 10 4 2 3 Optional CD8 enrichment 12 4 2 4 Isolation of antigen specific T cells with Streptamer Magnetic Beads protocol for human cells 14 4 2 5 Isolation of antigen specific T cells with Streptamer Magnetic Beads protocol for mouse cells 15 4 2 6 Magnetic separation on Miltenyi LS and MS columns 16 4 2 7 Magnetic separation with the autoMACS separator 17 4 2 8 Dissociat
10. ficiently removed by the addition of biotin This removal of the Strep Tactin backbone leaves monomeric Streptamer MHC class proteins on the surface of the T cell As the monovalent MHC I T cell receptor interaction is weak Streptamer MHC class proteins spontaneously dissociate from the T cell receptor and may be removed from the T cells simply by washing Keeping stained cells at 4 C together with rapid and complete dissociation of Streptamers from the T cells after purification assures the isolation of fully functional non induced T cells Further information is available at www streptamer com 4 Streptamer Manual Antigen specific staining and functional isolation of T cells 3 Streptamer Fluorescent T cell Labeling and Isolation via FACS 3 1 Introduction Scheme of a fluorescent Streptamer labeled T cell and biotin induced removal of the complex to yield a functional non induced antigen specific T cell preparation Peptide is Antigen Strep tag Biotin spontaneously MHC Peptide Antigen R i q q Strep tacg 9 3 2 Experimental procedure Routinely approximately 5x10 cells are stained using 0 75 ug Strep Tactin PE 5 ul and 1 Ug Streptamer MHC class 4 ul in a final volume of 50 ul Please note All steps the staining of the cells as well as the following dissociation of Streptamers have to be performed at 4 C Please make sure that all your reagents and the cells have reache
11. iba Solutions For Life Sciences Streptamer Manual Antigen specific staining and functional isolation of T cells Last date of revision April 2012 Version PR22 0005 www streptamer com For research use only Important licensing information Strep tag technology for protein purification and detection is covered by US patent 5 506 121 DE patent 42 37 113 JP patent 3865792 UK patent 2272698 and French patent 93 13 066 the tetracycline promoter based expression system is covered by US patent 5 849 576 and EU patent 759 997 and Strep Tactin is covered by US patent 6 103 493 and EU patent 835 934 Streptamer technology is covered by US Patent 7 776 562 and JP Patent 4 416 400 Further patent applications are pending world wide Purchase of reagents related to these technologies from IBA provides a license for non profit and in house research use only Expression or purification or other applications of above mentioned technologies for commercial use require a separate license from IBA A license may be granted by IBA on a case by case basis and is entirely at IBA s discretion Please contact IBA for further information on licenses for commercial use Trademark information i Strep tag Strep Tactin and Streptamer are registered trademarks of IBA GmbH Other trademarks and disclaimers MACS is a registered trademark of Miltenyi Biotec GmbH Ficoll Paque is a registered trademark of GE Healthcare Regist
12. ion of Streptamers with D biotin 17 4 2 9 Staining of T cells with Streptamers 17 5 Appendix Buffer Composition 18 6 References 19 Streptamer Manual Antigen specific staining and functional isolation of T cells 3 2 The Streptamer Principle Strep tag Strep Tactin and Streptamer Strep tags are short peptides with high binding selectivity for Strep Tactin an engineered Streptavidin The binding affinity of e g Strep tag Il to Strep Tactin Kd 1 uM is nearly 100 times higher than to streptavidin Strep tags may be fused to recombinant proteins which allows efficient one step purification of such fusion proteins on immobilized Strep Tactin under physiological conditions thus preserving their bioactivity As the Strep tag binds to the biotin binding pocket of Strep Tactin purified proteins may be mildly eluted from the column by the addition of minute amounts of biotin Further information is available at www strep tag com A special application of the Strep tag Strep Tactin technology is the oligomerization of MHC Strep tag fusion proteins Streptamer MHC class on Strep Tactin These complexes may be used for the efficient antigen specific staining of T cells by using moditied Strep Tactin being labeled by a fluorescent probe or a magnetic particle After separation of the stained T cells from non stained cells by FACS or by magnetic field separation respectively the staining or the magnetic particle may be ef
13. itive fraction outlet port pos1 This fraction represents the magnetically labeled non CD8 T cells Streptamer Manual Antigen specific staining and functional isolation of T cells 13 4 2 4 Isolation of antigen specific T cells with Streptamer Magnetic Beads protocol for human cells When working with anti coagulated peripheral blood or buffy coat PBMC should be isolated by density gradient centrifugation first see Protocol 4 2 2 This Protocol is adapted for 2 x 10 cells Higher cell numbers require larger amounts of beads and MHC 1 Thaw frozen cells in normal growth medium e Make sure concentration of DMSO is below 1 e Cells grown in medium containing less than 10 FCS should be thawed in medium containing 10 FCS instead of their normal growth medium 2 Wash cells in Buffer IS and resuspend in 10 ml use 300 g for each centrifugation 3 Pass cells through a nylon filter tube included in Streptamer Solution Set e This is necessary to remove cell clumps which may clog the columns 4 Determine cell number take sample before separation and place cells on ice 5 Incubate 50 pl Streptamer Magnetic Beads 8 pl Streptamer MHC class I and 90 ul Buffer IS at least 45 minutes at 4 C or over night 6 Place MiniMACS column in the magnetic field and prepare column by rinsing with 2 ml Buffer IS 7 Add 1 ml Buffer IS to MHC magnetic beads solution and load on MS column e
14. neg1 This fraction represents mostly T cells specific for other antigens and other cell types 4 2 8 Dissociation of Streptamers with D biotin 1 Centrifuge eluted cells and resuspend in 2 ml Buffer IS containing 1 mM D biotin and incubate for 20 minutes 2 Centrifuge cells and resuspend in 2 ml Buffer IS containing 1 mM D biotin and incubate for another 20 minutes 3 Wash cells 4 x with 5 ml Buffer IS 4 2 9 Staining of T cells with Streptamers To evaluate the purity of the enriched antigen specific T cells fractions can be analyzed by flow cytometry Live dead stain can be analyzed with ethidium monazid bromide EMA Molecular Probes E1374 CD8 T cells can be detected using a CD8 PE CD8 FITC antibody and optionally a T cell marker like CD3 can be detected with e g CD3 FITC CD3 APC antibody The antigen specitic fraction of these cells can be detected by using the same Streptamer MHC class in combination with a Strep Tactin PE conjugate Proceed as described under 3 2 2 When secondary staining of CD3 or CD8 is desired add the respective antibody 25 minutes after the addition of the Strep Tactin PE MHC complex to the cells so that its incubation will last 20 minutes total incubation of the Strep Tactin PE MHC with the cells 45 minutes Streptamer Manual Antigen specific staining and functional isolation of T cells 17 5 Appendix Buffer Composition Phosphate buffered saline PBS Buffer
15. ther applications Important All steps have to be performed at 4 C Streptamer Manual Antigen specific staining and functional isolation of T cells 4 Streptamer Magnetic T cell Labeling and Isolation via MACS 4 1 Introduction 4 1 1 Purification scheme In a first step T cells are labeled with a magnetic Streptamer complex according to their antigen speciticity then stained T cells are separated from other cells by a magnetic field and such purified T cells are eluted and released from the Streptamer complex by the addition of biotin vitamin H to yield a functional non induced antigen specific T cell preparation labeling loading amp washing elution dissociation T cells of other specificities T cell of _ interest TCR QUQ peptide antigen Mine T Strep tag J B aana i Strep Tactin magnetic bead J spontaneously i T cell of interest 4 1 2 Recommendations for isolating T cells using Streptamer Magnetic Beads and recombinant Streptamer MHC class Experimental procedure The procedure is optimized to isolate antigen specific T cells from 2x10 peripheral blood mononuclear cells PBMC Some cells like monocytes or natural killer cells may also be co puritied due to their ability to bind MHC and can be depleted before the actual T cell isolation The recommended procedure depends on species and source of cells For human blood e Anticoagulant trea
16. tment 4 2 1 e Ficoll gradient 4 2 2 e T cell isolation 4 2 4 Streptamer Manual Antigen specitic staining and functional isolation of T cells 7 For mouse blood Anticoagulant treatment 4 2 1 Ficoll gradient 4 2 2 CD8 enrichment 4 2 3 Optional NK cell depletion Antigen specitic T cell isolation 4 2 5 Please note All steps the isolation of cells as well as the following dissociation of Streptamers have to be performed at 4 C Please make sure that all your reagents and the cells have reached the temperature before starting the protocol 4 1 3 Example of an isolation of CMV antigen specific T cells Isolation of antigen specific T cells from PBMC using Streptamer Magnetic Beads Cat No 6 5500 005 and recombinant Streptamer MHC class HLA A2 CMV PP65 Cat No 6 7001 005 Streptamer Magnetic Bead sorting before sort after sort 0 079 MHC Multimer CD8 human PBMCs CMYV MHC HLA A2 pp6549 lt 593 F Anderl et al unpublished data 8 Streptamer Manual Antigen specific staining and functional isolation of T cells 4 1 4 Set of reagents for isolation of antigen specific T cells ltem Cat No Quantity Streptamer Magnetic Beads 6 5500 005 250 ul suff for 1 10 cells Streptamer Magnetic Beads 6 5500 025 1 25 ml suff for 5 10 cells Streptamer MHC class 6 70xx 005 200 ul suff for pur of 5 10 human or 2 5 10 mous
17. umns 3 Determine cell number take sample before separation approximately 1 5 x 10 cells are required per staining and place cells on ice 4 Incubate 50 pl Streptamer Magnetic Beads and 16 pl Streptamer MHC class and 80 pl Buffer IS at least 45 minutes at 4 C or over night 5 Place MiniMACS column in the magnetic field and prepare column by rinsing with 2 ml Buffer IS 6 Add 1 ml Buffer IS to MHC magnetic beads solution and load on MS column To wash away unbound MHC magnetic beads are bound and washed on a MS column 7 Wash with 2 ml Buffer IS 8 Add 250 wl Buffer IS and elute retained beads outside of the magnetic field into a fresh vial and firmly flush out the beads using the supplied plunger supplied with the column 9 Centrifuge cell suspension and resuspend in 250 ul magnetic beads MHC solution Incubate 45 minutes on ice 10 Add 1 5 ml Buffer IS centrifuge cell and beads mixture 10 minutes at 300 x g at 4 C and wash once by resuspending in 2 ml Buffer IS and centrifuging as above This is necessary to eliminate unbound magnetic beads which may trap cells on the column un specifically 11 Resuspend in 2 ml Buffer IS Proceed to magnetic separation Streptamer Manual Antigen specific staining and functional isolation of T cells 15 4 2 6 Magnetic separation on Miltenyi LS and MS columns For best results the separation is repeated on a LS and MS column For detailed description
18. utic purposes A comparative study CID 46 e96 105 Streptamer Manual Antigen specific staining and functional isolation of T cells 19 Solutions For Life Sciences IBA Headquarters IBA IBA GmbH Rudolf Wissell Str 28 37079 Goettingen Germany Tel 49 0 551 50672 0 Fax 49 0 551 50672 181 E mail info iba lifesciences com US Distribution Center 1328 Ashby Road Olivette MO 63132 USA Tel 1 877 IBA GmbH 1 877 422 4624 Fax 1 888 531 6813 E mail info iba lifesciences com
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