Manual_MHC_Streptamer
Contents
1. 3 4 7 Short Protocol Please request a copy of our Short Protocol PR36 Human or PR37 Mouse for MHC Streptamer Isolation at info streptamer com or download it from http www iba lifesciences com technical support html MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 13 4 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 Neudorfer 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 Harter 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 phosphopr2. amount of Streptamers stepwise 2 3 and 4 fold Add the following volumes of pre incubated 45 min Streptamers to your cell pellet e 2 fold increase 8 ul MHC I Strep 10 ul Strep Tactin PE in 100 ul buffer IS e 3 fold increase 12 ul MHC I Strep 15 ul Strep Tactin PE in 150 ul buffer IS e 4 fold increase 16 ul MHC I Strep 20 ul Strep Tactin PE in 200 ul buffer IS The assay can be conducted in a 96 well round bottom microplate 2 6 Dissociation of Streptamers with D Biotin Important All steps 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 1 Collect cells by centrifugation 2 Prepare ca 1 ml Buffer IS containing 1 mM D Biotin 1 mM Biotin working solution 3 Resuspend cells in 200 ul Biotin working solution and incubate for 10 minutes 4 Collect cells by centrifugation 5 Repeat step 3 and 4 6 Wash cells 4 times with 200 ul Buffer IS 7 Transfer cells into the appropriate buffer or medium for further applications 2 7 Short Protocol Please request a copy of our Short Protocol PR38 for MHC Streptamer Staining at info streptamer com or download it from www streptamer com 8 MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 3 Magnetic isolation of antigen specific CD8 T cells with MHC I Streptamers 3 1 The principle magnetic isolation and removal of l
3. at 4 C or overnight 4 Proceed to 3 4 2 3 4 2 Cell Labeling with Streptamers Purification of Streptamers removal of unbound MHC I Strep step 1 4 1 Place MS column in the magnetic field and prepare column by rinsing with 2 ml Buffer IS 2 Add 1 ml Buffer IS to Streptamers from 3 4 1 step 3 human or mouse and load on MS column 3 Wash MS column while in magnetic field with 2 ml Buffer IS to remove unbound MHC I Streps 4 Add 250 ul Buffer IS to MS column and elute retained Streptamers beads with bound MHC I Streps outside the magnetic field into a fresh vial firmly flush out the purified Streptamers using the plunger supplied with the column Cell Labeling with purified Streptamers step 5 7 5 Centrifuge cell suspension 300 x g and resuspend the cells in 250 ul purified Streptamers from 3 4 2 step 4 Incubate 45 minutes on ice 6 Add 1 5 ml Buffer IS centrifuge cell and Streptamer mixture and carefully wash with 2 ml Buffer IS to eliminate unbound magnetic beads which may trap cells on the column unspecifically 7 Resuspend cells in 2 ml Buffer IS Proceed to 3 4 3 or 3 4 4 12 MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 3 4 3 Magnetic separation with LS column 8 Place LS column in the magnetic field and prepare column by rinsing with 3 ml Buffer IS 9 Apply cell suspension 3 4 2 step 7 onto the column Allow unlabeled cells to pass thr
4. 0 005 Strep Tactin APC for MHCI Streptamers 2 5x10 cells 0 25 ml 2 2 2 Streptamer Solution Set and pre separation filters Cat no Product Name Content 6 5603 005 Streptamer Solution Set Standard Buffer IS D Biotin The Streptamer Solution Set Standard contains 50 ml Buffer IS as 10x concentrate for washing and 1 ml of a D Biotin stock solution 100 mM for dissociation of the Streptamers from the isolated cells Buffer IS has to be diluted with 9 volumes of water prior to use We recommend to add EDTA at a final concentration of 1 mM Degas buffer before use The 100 mM Biotin stock solution has to be diluted with 99 volumes of Buffer IS prior to use Biotin working solution is 1 mM see 2 6 Pre separation filters IBA GmbH cat no 6 5601 010 are recommended for removal of cell clumps 6 MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 2 3 Use and storage of MHC I Streps and fluorescent Strep Tactin MHC I Streps are shipped on dry ice and then stored at 80 C until use After initial thawing prepare aliquots for long term storage at 80 C Aliqouts for immediate use should be kept permanently on ice Aliquotation is mandatory to avoid freeze thaw cycles which denature the MHC I Streps Strep Tactin PE or APC for MHC I Streptamers is shipped on blue ice and stored at 4 C 2 4 Staining procedure The procedure is optimized for staining of antigen specific CD8 T cel
5. HC I Strep proteins on the surface of the T cell As the interaction of monovalent MHC I T cell receptor is weak MHC I Strep proteins spontaneously dissociate from the T cell receptor and may be removed from the T cells simply by washing Keeping cells at cooled conditions as well as performing the rapid and complete removal of the Streptamers from the T cells assures the isolation of fully functional non induced T cells Further information is available at www streptamer com MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 5 2 Reversible staining of antigen specific CD8 T cells with MHC I Streptamers and FACS isolation 2 1 Introduction cell staining and removal of staining reagents Scheme of a fluorescent Streptamer labeled T cell and subsequent biotin induced removal of the Streptamers to yield a functional non induced antigen specific T cell preparation specific labeling reagent dissociation amp removal T cells of other specificity D biotin T cell of ZA interest o spontaneously g TCR i gt MHC peptide antigen functional and label free Strep tag Strep tag T cell of interest T cell of G interest fluorochrome AN 2 2 Required reagents and materials 2 2 1 MHC l Streps and Strep Tactin PE or APC Cat no Product Name Staining of Size 6 7XXX 005 MHC l Strep 2 5x10 cells 0 2 ml 6 5000 005 Strep Tactin PE for MHC I Streptamers 2 5x10 cells 0 25 ml 6 501
6. abeling reagents CD8 T cells are labeled according to their antigen specificity with Strep Tactin Magnetic Nanobeads coupled to the specific MHC I Strep Labeled cells are separated from other cells by a magnetic field and retained target cells are eluted after removal of the magnet All Streptamer reagents are then released from the target cells by the addition of biotin vitamin H to yield a functional non induced antigen specific CD8 T cell preparation specific labeling washing elution reagent dissociation amp removal T cells of other specificity magnet column Ss Strep tag D biotin D biotin vere interest T cell of 4 J spontaneously Y peptide antigen Strep tag Strep Tactin magnetic nanobead T cells of other specificit p Y Strep Tactin functional and label free T cell of interest Example Isolation of CMV antigen specific T cells Isolation of human antigen specific T cells from PBMC with MHC Streptamers Antigen specific T cells were positive selected with Strep Tactin Magnetic Nanobeads for MHC Streptamers which were coupled to MHC I Strep HLA A 0201 CMV pp6549s5 503 Magnetically labeled cells were isolated on a MACS column Thereafter the Streptamer reagents were removed from the target cells with 1 mM D Biotin and cells were analyzed by flow cytometry Before sort the antigen specific T cells represented only 0 079 of the total cell population Afte
7. before starting the protocol Avoid foaming which interferes with proper bead retention on the magnet 3 4 1 Preparation of cells and Streptamers Human cells The procedure is optimized to isolate antigen specific CD8 T cells from 2 x10 freshly isolated or frozen peripheral blood mononuclear cells PBMCs When working with anti coagulated peripheral blood or buffy coat PBMCs should be isolated by density gradient centrifugation first Mouse cells When working with cells from spleen or lymph node 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 I Streps MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 11 Cell preparation step 1 2 1 Collect prepared cells and resuspend in 10 ml Buffer IS 2 Pass cells through a nylon filter tube to remove cell clumps which may clog the columns and place cells on ice MHC I Streptamer preparation for human cells step 3 3 Incubate 50 ul Strep Tactin Magnetic Nanobeads 8 ul MHC I Strep and 90 ul Buffer IS at least 45 minutes at 4 C or overnight 4 Proceed to 3 4 2 MHC I Streptamer preparation for mouse cells step 3 3 Incubate 50 ul Strep Tactin Magnetic Nanobeads 16 ul MHC I Strep and 80 ul Buffer IS at least 45 minutes
8. iba Solutions For Life Sciences MHC I Streptamer Manual I Staining of antigen specific CD8 T cells with reversible MHC Streptamers and FACS isolation Il Isolation of antigen specific CD8 T cells with reversible MHC Streps and Strep Tactin Magnetic Nanobeads Last date of revision July 2015 Version PR22 0012 For research use only Important licensing information The technologies Streptamer and Strep Tactin are covered by intellectual property IP rights On completion of the sale of a respective product IBA grants a Limited Use Label License to purchaser IP rights and Limited Use Label Licenses are further identified at http www iba go com patents html or upon inquiry at info iba go com or at IBA GmbH Rudolf Wissell Str 28 37079 Gottingen Germany By use of a respective product the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses All products are for research use only CAUTION Not intended for human or animal diagnostic or therapeutic uses Trademark information The owners of trademarks marked by or TM are identified at http www iba go com patents html Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law 2 MHC I Streptamer Manual Staining and isolation of antigen specific CD8 T cells Content 1 The Streptamer Principle 5 2 Rever
9. ion of antigen specific CD8 T cells 3 2 3 Magnetic columns and pre separation filters For magnetic separation we recommend the MS or LS columns with the MACS Manual Separators or the AutoMACS from Miltenyi Biotech GmbH For removal of cell clumps prior to column loading we recommend our pre separation nylon filters IBA GmbH cat no 6 5601 010 includes 10 filters 3 3 Use and storage of MHC I Streps and magnetic beads MHC I Streps are shipped on dry ice and then stored at 80 C until use After initial thawing prepare aliquots for long term storage at 80 C Aliqouts for immediate use should be kept permanently on ice Aliquotation is mandatory to avoid freeze thaw cycles which denature the MHC I Streps Strep Tactin Magnetic Nanobeads for MHC Streptamers are shipped on blue ice and stored at 4 C do not freeze 3 4 Experimental procedure The procedure is optimized for T cell isolation from 2 x10 cells For cell numbers higher than 2 x10 we suggest a linear upscale of beads and MHC I Streps Some cells like monocytes or natural killer cells may be co purified due to their ability to bind MHC I they can be depleted by density gradient centrifugation or CD8 pre selection prior to T cell isolation Important 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
10. ls from fresh or frozen peripheral blood mononuclear cells PBMCs When working with anti coagulated peripheral blood or buffy coats PBMCs should first be isolated by density gradient centrifugation and separated from platelets Please adjust cell density to 10 cells 100 pl before starting the protocol Important All steps 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 Protect labeled cells and fluorochrome reagents from light by incubating in the dark Protocol for staining of ca 5x10 cells 1 test 1 Prepare ca 3 ml Buffer IS from 10 x stock 2 Incubate 5 ul Strep Tactin PE or APC and 4 ul MHC I Strep in a final volume of 50 ul Buffer IS for 45 minutes 3 Add the pre incubated Streptamers complex from Strep Tactin PE or APC and MHC I Strep step 2 to the cell pellet 4 Incubate for 45 minutes 5 Wash cells twice with 200 ul Buffer IS 6 Cells are ready for FACS analysis or FACS sorting Dead cell exclusion is strongly recommended e g propidium iodide 7 AAD etc MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 7 2 5 Titration optional If the staining protocol is not suitable for your application a titration of the Streptamers should be performed Our recommendation for the titration is Keep the cell concentration of 10 cells 100 ul constant and increase the
11. otein 65 specific T cells for diagnosis and therapeutic purposes A comparative study CID 46 e96 105 For more references please visit www streptamer com 5 Warranty The product sold hereunder is warranted only to conform to the quantity and contents stated on the label at the time of delivery to the customer There are no warranties expressed or implied that extend beyond the description on the label of the product IBA s sole liability is limited to either replacement of the products or refund of the purchase price Iba GmbH is not liable for property damage personal injury or economic loss caused by the product Please refer to www iba lifesciences com technical support html for downloading this manual 14 MHC I Streptamer Manual Staining and isolation of antigen specific CD8 T cells Solutions For Life Sciences IBA Headquarters 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 IBA 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
12. ough 10 Wash column with 3 x 3 ml Buffer IS adding buffer each time once the column reservoir is empty 11 Remove column from magnetic field add 5 ml Buffer IS and flush out labeled cells positive fraction into a fresh vial by firmly applying the plunger supplied with the column To increase purity the magnetically labeled fraction can be passed over a new MS column for up to 10 labeled cells or LS column for up to 10 labeled cells 3 4 4 Optional Magnetic separation with the AutoMACS separator For detailed instructions on how to use the AutoMACS please refer to the corresponding user manual Choose program Posseld and collect positive cell fraction 3 4 5 Dissociation of Streptamers with D Biotin 12 Centrifuge eluted cells positive fraction resuspend in 2 ml Buffer IS containing 1 mM D Biotin and incubate for 10 minutes 13 Repeat step 12 14 Wash cells 3 times with 5 ml Buffer IS 3 4 6 Staining of T cells with Streptamers Antigen specific cells can be visualized by utilizing the same MHC I Strep which was used for magnetic isolation in combination with Strep Tactin PE or APC Proceed as described under 2 4 When a combinatorial staining with antibodies especially anti CD3 or anti CD8 mAbs is desired add the respective antibodies only for the last 20 min of the staining protocol 2 4 in total 45min to avoid interference with the Streptamers A live dead discrimination is suggested
13. r sort an enormous enrichment by a factor of 1 000 and a high purity of 82 became evident These two important aspects were achieved with positive selection in only one step MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 9 before sort after sort 0 079 MHC Multimer CD8 human PBMCs CMV MHC HLA A2 pp65 496 593 3 2 Streptamer reagents and magnetic columns for cell isolation 3 2 1 MHC I Streps and Strep Tactin Magnetic Nanobeads Cat no Product Name Isolation from Size 6 7XXX 005 MHC I Strep 5 x 10 cells human 1x0 2 ml 2 5 x10 cells mouse 6 5500 005 Strep Tactin Magnetic Nanobeads 8 for MHC Streptamers 1 x 10 cells 1x 0 25 ml 3 2 2 Streptamer Solution Set Standard for washing amp dissociation Cat no Product Name Content 6 5603 005 Streptamer Solution Set Standard Buffer IS D Biotin The Streptamer Solution Set Standard contains 50 ml Buffer IS as 10x concentrate for washing and 1 ml of a D Biotin stock solution 100 mM for dissociation of the Streptamers from the isolated cells Buffer IS has to be diluted with 9 volumes of water prior to use We recommend to add EDTA at a final concentration of 1 mM Degas buffer before use as air bubbles may block the column The 100 mM Biotin stock solution has to be diluted with 99 volumes of Buffer IS prior to use Biotin working solution is 1 mM 10 MHC Streptamer Manual Staining and isolat
14. sible staining of antigen specific CD8 T cells with MHC I Streptamers and FACS isolation 6 2 1 Introduction cell staining and removal of staining reagents 6 2 2 Required reagents and materials 6 2 2 1 MHC I Streps and Strep Tactin PE or APC 6 2 2 2 Streptamer Solution Set and pre separation filters 6 2 3 Use and storage of MHC I Streps and fluorescent Strep Tactin 7 2 4 Staining procedure 7 2 5 Titration optional 8 2 6 Dissociation of Streptamers with D Biotin 8 2 7 Short Protocol 8 3 Magnetic isolation of antigen specific CD8 T cells with MHC I Streptamers 9 3 1 The principle magnetic isolation and removal of labeling reagents 9 3 2 Streptamer reagents and magnetic columns for cell isolation 10 3 2 1 MHC I Streps and Strep Tactin Magnetic Nanobeads 10 3 2 2 Streptamer Solution Set Standard for washing amp dissociation 10 3 2 3 Magnetic columns and pre separation filters 11 3 3 Use and storage of MHC I Streps and magnetic beads 11 3 4 Experimental procedure 11 3 4 1 Preparation of cells and Streptamers 11 3 4 2 Cell Labeling with Streptamers 12 3 4 3 Magnetic separation with LS column 13 3 4 4 Optional Magnetic separation with the AutoMACS separator 13 3 4 5 Dissociation of Streptamers with D Biotin 13 3 4 6 Staining of T cells with Streptamers 13 3 4 7 Short Protocol 13 4 References 14 5 Warranty 14 MHC Streptamer Manual Staining and isolation of antigen specific CD8 T cells 3 MHC I S
15. treptamer Manual Staining and isolation of antigen specific CD8 T cells e e 1 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 MHC I Strep proteins on Strep Tactin Multimers of MHC I Streps complexed with either fluorescently or magnetically labeled Strep Tactin so called Streptamers are used for efficient staining or isolation of antigen specific T cells After separation of the labeled T cells from non labeled cells by flow cytometric or magnetic cell isolation the Streptamers are efficiently disrupted on the cell by addition of biotin Subsequently the dissociation and removal of the Strep Tactin backbone leaves monomeric M
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