Fab Streptamer Microbeads Manual, human

Transcription

1 Fab Streptamer Microbeads Manual, human Isolation of functional target cells with reversible Fab-Streps and Strep-Tactin Magnetic Microbeads Last date of revision August 2016 Version PR

2 For research use only Important licensing information The technologies Streptamer and Strep-Tactin described 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 or upon inquiry at info@iba-go.com or at IBA GmbH, Rudolf-Wissell-Str. 28, Göttingen, 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 Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

3 Content 1 The Streptamer Principle 4 2 Fab Streptamer Magnetic Cell Labeling and Cell Isolation with Strep-Tactin Magnetic Microbeads on a permanent magnet Introduction Purification Scheme Example of CD8 + target cell enrichment from human PBMCs Streptamer reagents Fab Streptamer Isolation Kits MB: cell isolation with magnetic microbeads Pre-separation filters StrepMan Magnet Use and storage of Fab-Streps and magnetic microbeads Experimental procedure Purification of CD8 + (T) cells from 1x10 7 human PBMCs with CD8 Fab-Strep and Strep-Tactin Magnetic Microbeads Dissociation of Fab Streptamers from the isolated cell population Titration (optional) Short Protocol 11 3 Warranty 11

4 Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 2

5 Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 3

6 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 II to Strep-Tactin (K d = 1 µm) 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 Strep-tags bind 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. A special application of the Strep-tag : Strep-Tactin technology is the oligomerization of Fab-fragment-Strep-tag fusion proteins (Fab-Strep proteins) on Strep-Tactin. Multimers of Fab-Streps complexed with either fluorescently or magnetically labeled Strep-Tactin, socalled Streptamers, are used for efficient staining or isolation of cells. After separation of the labeled 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 Fab-Strep proteins on the surface of the cell. As the interaction of monovalent Fab : cell receptor is weak, Fab-Streps spontaneously dissociate from the cell receptor and may be removed from the cells simply by washing. Keeping cells at cooled conditions as well as performing the rapid and complete removal of the Streptamers from the cells assures the isolation of fully functional, non-induced cells. Further information is available at Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 4

7 2 Fab Streptamer Magnetic Cell Labeling and Cell Isolation with Strep-Tactin Magnetic Microbeads on a permanent magnet 2.1 Introduction Purification Scheme Receptor specific target cells are first labeled with magnetic Streptamers, i.e. paramagnetic Fab-Strep : microbead complexes, and subsequently separated from other cells by positive selection in a strong permanent magnetic field. In a second step, the isolated cells are liberated from all labeling reagents by the addition of D-biotin (vitamin H) and washing in order to obtain a functional, completely label-free target cell preparation. The Fab-Strep ligands are engineered to have week affinity to the cognate cell surface receptor and the monomeric ligand therefore can be rapidly removed from its receptor by simple washing steps Example of CD8 + target cell enrichment from human PBMCs Isolation of CD8 + cells from human PBMCs using recombinant CD8 Fab-Strep multimerized on Strep-Tactin Magnetic Microbeads. live (PI negative ) cells are shown Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 5

8 2.2 Streptamer reagents Fab Streptamer Isolation Kits MB: cell isolation with magnetic microbeads Cat.no. Product Name Amount 6-80XX-2XX 6-85XX-2XX CDX Fab Streptamer Isolation Kit MB, human for isolation out of 1x10 9 cells CDX Fab Streptamer Isolation Kit MB, mouse for isolation out of 2x10 9 or 1x10 9 cells The Fab Streptamer Isolation Kits MB contain Fab-Streps and Strep-Tactin magnetic microbeads (MB) for manual cell isolation with a permanent magnet (e.g. IBA s StrepMan). The kits furthermore contain Buffer IS as 10x concentrate for washing, and 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 2.4.2) Pre-separation filters We recommend our pre-separation nylon filters for removal of cell clumps (cat.-no.: ; includes 10 filters) StrepMan Magnet Cat.no. Product Name Size StrepMan Magnet for 2 x 15 ml and 2 x 50 ml tubes a b The StrepMan magnet can be used in two different positions. For separation of the magnetically labeled cells (2.4.1), we recommend a horizontal position (a); for removal of the magnetic Streptamers from the isolated cells (2.4.2) we recommend an upright position (b). Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 6

9 2.3 Use and storage of Fab-Streps and magnetic microbeads Fab-Streps are shipped on dry ice and then stored at -80 C until use. After thawing keep them on ice during use. Residual amounts after initial use should be aliquoted and stored at - 20 C. Aliquotation is mandatory to avoid freeze thaw cycles, which denature the Fab-Streps. Strep-Tactin Magnetic Microbeads for Fab Streptamers are shipped on blue ice and stored at 4 C (do not freeze). The storage buffer of the beads contains 0.05 % sodium azide. We therefore recommend to wash the beads with 1 volume of buffer IS (or at least 1 ml) on the magnet prior to use. 2.4 Experimental procedure The procedure is optimized for the isolation of CD8 + (T) cells from up to 1x10 7 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. In order to obtain best purities and yields, especially when targeting very large populations (like CD3 + cells) you may need to optimize the amount of selection reagents. The protocol can be scaled up for target cell isolation from larger cell numbers, please refer to tables 1 and 2 for starting point suggestions. Please note: Purification of cells (2.4.1) has to be performed at 4 C. Please make sure that all reagents and cells are accordingly refrigerated before starting the protocol. The subsequent dissociation of Fab Streptamers and washing (2.4.2) is performed at room temperature Purification of CD8 + (T) cells from 1x10 7 human PBMCs with CD8 Fab-Strep and Strep-Tactin Magnetic Microbeads Avoid foaming, which interferes with proper bead retention on the magnet! Please perform at 4 C (please refer to highlighted notes above). Fab Streptamer preparation (step 1 and 2): Optional: Wash magnetic microbeads before use to remove sodium azide. Therefor transfer the appropriate amount of homogeneously resuspended Strep-Tactin Magnetic Microbeads to a new tube. Add 1 ml of buffer IS and resuspend carefully by pipetting up and down. Hold the tube on the magnet for at least 1 min and discard the supernatant. Remove the tube from the magnet and resuspend the Strep-Tactin Magnetic Microbeads in the appropriate initial volume of fresh buffer IS. Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 7

10 1. Mix 4 µl Fab-Strep with 1 µl Buffer IS in a fresh reaction tube. 2. Add 15 µl from the homogeneously resuspended Strep-Tactin Magnetic Microbeads to the Fab-Strep solution above. Ensure proper mixing to achieve a homogeneous Fab- Strep distribution on the Strep-Tactin Magnetic Microbeads. Incubate under gentle constant agitation, e.g. on a roller mixer, for 30 minutes (overnight incubation might be suitable). Cell labeling with Fab Streptamers (step 3-5): 3. OPTIONAL: Wash up to 1x10 7 pre-cooled cells with 10 ml Buffer IS in a 15 ml reaction tube to remove potentially interfering components (e.g. D-biotin). Resuspend the cells in 30 µl Buffer IS. 4. Add the pre-incubated Fab-Strep Microbead preparation (magnetic Streptamers ) from step 2 to the cells and mix thoroughly by gentle pipetting. 5. Incubate for 20 minutes under gentle constant agitation, e.g. on a roller mixer, to prevent cells from sedimentation. Magnetic separation (step 6-9): 6. Add 5 ml of buffer IS to the cell:bead preparation from step Separate magnetically labeled cells by placing the tube for at least 3 min firmly onto the StrepMan Magnet. For best results we suggest to tilt the magnet horizontally. Remove the supernatant containing the negative cell fraction by careful pipetting while keeping the reaction tube on the StrepMan Magnet. 8. Remove the reaction tube from the StrepMan Magnet and carefully flush labeled cells (positive cell fraction) off the tube wall by resuspending in 5 ml Buffer IS. 9. Repeat magnetic selection (steps 7 and 8) twice. 10. Finally resuspend labeled cells (positive cell fraction) as described under step 2 Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 8

11 Table 1: Fab Streptamer labeling Suggestions for adaptation of reagent amounts to different cell numbers pre-sort cell number Fab- Strep Buffer IS microbeads tube size cell volume final volume washing (StrepMan Magnet) Step 1 Step 1 Step 2 Step 3 Step 3 Step 4 Step 6 1x µl 1 µl 15 µl 15 ml 30 µl 50 µl 5 ml 1x µl 10 µl 150 µl 15 ml 300 µl 500 µl 10 ml 1x µl 100 µl 1.5 ml 50 ml 3 ml 5 ml 45 ml Dissociation of Fab Streptamers from the isolated cell population The following protocol describes the removal of Streptamer reagents from 1x10 7 cells. Dissociation/washing steps in larger volumes are generally possible for the isolation of more abundant cell fractions or from higher cell numbers, please refer to Table 2. All steps in are performed at room temperature. Dissociation and removal of Strep-Tactin Magnetic Microbeads (step 1-6) 1. Prepare a 1 mm D-biotin working solution by mixing 10 ml buffer IS and 100 µl 100 mm D-biotin stock solution. 2. Resuspend final cell fraction from step 10, , in 5 ml 1mM D-biotin working solution and mix thoroughly by pipetting. Incubate for 10 minutes. 3. Place the reaction tube on the StrepMan Magnet for at least 3 minutes. Carefully pipet off the supernatant containing the target cell fraction and transfer the supernatant to a fresh reaction tube. 4. Remove the reaction tube from the StrepMan Magnet and thoroughly resuspend residual cells and magnetic beads with 5 ml 1 mm D-biotin working solution. Incubate for 10 minutes. 5. Repeat step Pool the supernatants and collect cells by centrifugation (400 g, 6-10 min). Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 9

12 Removal of Fab-Streps (step 7-10) 7. Carefully remove supernatant and discard. Resuspend cell pellet in 5 ml Buffer IS and incubate for 10 minutes under agitation at room temperature. 8. Place tube back on the magnet (to remove any potential residual beads) and incubate for 3 minutes. 9. After incubation, transfer supernatant to a new tube and collect cells in this tube by centrifugation (400 x g, 6-10 min). 10. Repeat step 7 and 8 and pool cell fractions. Collect cells by centrifugation (400 x g, 6-10 min). 11. Remove supernatant and resuspend cells in the appropriate buffer or medium for further applications. To perform a further positive isolation please start the protocol once more at Table 2: Dissociation of Fab Streptamers Suggestions for adaptation of reagent amounts to different cell numbers post-sort cell number tube size biotin working solution dissociation bead removal Fab-Strep removal Step 1 Step 2 Step 4 Step 7 1x ml 10 ml 5 ml 5 ml 5 ml 1x ml 20 ml 10 ml 10 ml 10 ml 5x ml 30 ml 15 ml 15 ml 15 ml Titration (optional) If the protocol does not provide satisfactory results in a given application, especially for obtaining high yields of very abundant target cell populations, a titration of the ratio between the Streptamers (Fab-Strep microbead preparation) and the cell number should be performed. Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 10

13 2.4.4 Short Protocol Please request a copy of our Short Protocol PR40 for Fab Streptamer Microbeads at info@streptamer.com or download it from 3 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. Fab-Streptamer Microbeads Manual cell enrichment with Strep-Tactin Magnetic Microbeads 11

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