“Do-It-Yourself” Selection Kit
Contents
1. Note This procedure is designed for use with the EasySep Magnet 18000 If using The Big Easy EasySep Magnet 18001 please refer to www stemcell com technical 18001 PIS pdf for additional instructions If using RoboSep The Fully Automated Cell Separator please select the appropriate program or refer to the RoboSep user manual after completing Part A Cocktail Assembly and Part B Step 1 EasySep Procedure Add EasySep F component A and mix gt a Tube containing 15 ug a mouse IgG antibody a Add EasySep component B and mix EasySep Incubate at 37 C 5 hours to overnight Bring to a final volume of 1 mL and transfer to provided RoboSep compatible vial if needed EasySep Positive Selection Cocktail is now ready for use in Part B Part B B Add EasySep positive selection cocktail from Part A to single cell suspension 2 Cell suspension Incubate 15 minutes Add EasySep magnetic i nanoparticles Incubate 10 minutes Place tube in magnet for Pour off supernatant 5 minutes Positively selected cells remain in tube After two rinses collect cells simply by removing tube sfe Positive Selection Do It Yourself Selection Kit sEasySep CATALOG 18098 Version 2 0 1 The EasySep Do It Yourself Selection Kit contains all of the necessary components to create a specific EasySep positive selection cockt2. 2 mL of cells add 100 uL of nanoparticles Mix well and incubate at room temperature 10 minutes ube by gently pipetting up and down 2 3 times Place the tube without cap into the magnet Set asi or 5 minutes increasing separation time to 6 10 minutes may increase recovery see Notes and Tips and down more than 5 times Vortexing is not recommended Add nanoparticles at 50 pL mL cells e Bring cell suspension to a total volume of 2 5 mL by adding recommended medium Mix the cells in the nd Add the positive selection cocktail that has been assembled in Part A to the cell suspension at 100 pL mL or up g or ide Ji supernatan jeld of the EasySep Magnet Leave the magnet and tube inverted for 2 3 seconds then return upright position Do not shake or blot off any drops that may remain hanging from the mouth of the tube Remove the tube from the magnet and add 2 5 mL of recommended medium Mix the cell suspensi by gently pipetting up and down 2 3 times Place the tube back in the magnet and set aside for 5 minutes ube from magnet and resuspend cells in an appropriate amount of desired medium The positiv selected cells are now ready for use he to on Repeat Steps 6 and 7 and then Step 6 once more for a total of three 5 minute separations in the magnet for rare cells additional rounds of separation may improve purity see Notes and Tips Remove ely Users of this selection
3. cell type is to be selected from other sources tissues ensure that cells are in a single cell suspension for optimal results Recommended Medium The recommended medium is PBS with 2 FBS Catalog 07905 and 1 mM EDTA Medium should be Ca and Mg free Optimizing Purity For samples with a desired cell starting frequency of less than 10 15 additional separation rounds will likely improve purity If desired repeat Steps 6 and 7 an additional one to three times Please note that recovery will decrease with each additional round of separation Also for rare cells i e cells representing less than 5 of the initial population increasing initial cell concentration from 1 x 10 cells mL to 2 x 10 cells mL may improve purity Optimizing Recovery Recovery may be improved by increasing separation time in the magnet from 5 to 10 minutes for each round Recovery of positively selected cells is also dependent on the quality of the mouse IgG antibody used in the cocktail created with the EasySep Do It Yourself Selection Kit Antibodies that have expired or that have been stored improperly may show lower affinity for the surface marker on the target cell resulting in lower recovery Assessing Purity The mouse IgG antibody used in the cocktail created with the EasySep Do It Yourself Selection Kit may block other fluorochrome conjugated antibodies that are used to assess purity by flow cytometry We recomme
4. kit should ensure that they are entitled to use the antibody of interest StemCell Technologies is not responsible for patent infringements or violations that may occur when using this product StemCell Technologies In the United Kingdom Tel 44 0 20 7691 3561 Fax 33 0 4 76 18 99 63 Toll Free within United Kingdom Tel 0800 731 27 14 Fax 0800 731 27 13 e mail info stemcellgb com In North America Tel 1 604 877 0713 Fax 1 604 877 0704 Toll Free Tel 1 800 667 0322 Toll Free Fax 1 800 567 2899 e mail info stemcell com www stemcell com In Europe Tel 33 0 4 76 04 75 30 Fax 33 0 4 76 18 99 63 e mail info stemcellfrance com May 2007 28863 ee Printed on recycled paper FOR RESEARCH USE ONLY Catalog 18098 Components e EasySep Do It Yourself Component A e EasySep Do It Yourself Component B e EasySep Magnetic Nanoparticles e RoboSep Vial for Do It Yourself Antibody Cocktail not required for manual use For labeling 10 total cells EasySep 0 1 mL A 7 Positive Selection Um 1 vial Product Information Sheet REQUIRED EQUIPMENT EasySep Magnet Catalog 18000 or The Big Easy EasySep Magnet Catalog 18001 or RoboSep Catalog 20000 PRODUCT DESCRIPTION AND APPLICATIONS EasySep Do It Yourself Selection Components A and B create bispecific Tetrameric Antibody Complexes TAC when mixed together wi
5. Do It Yourself Antibody Cocktail code 18093 Empty vial compatible with RoboSep the fully automated cell separator If using RoboSep transfer the antibody cocktail assembled in Part A into this empty vial before loading into the appropriate position in the RoboSep carousel If not using RoboSep this vial can be discarded STABILITY AND STORAGE EasySep Do It Yourself Components A and B Stable at 4 C for 1 year Do not freeze these products Contents sterile in unopened tube EasySep Magnetic Nanoparticles Stable at 4 C for 2 years Contents sterile in unopened tube This product may be shipped at room temperature and should be refrigerated upon receipt Ficoll and Ficoll Paque PLUS are trademarks of GE Healthcare Ltd StemCell Technologies In the United Kingdom Tel 44 0 20 7691 3561 Fax 33 0 4 76 18 99 63 Toll Free within United Kingdom Tel 0800 731 27 14 Fax 0800 731 27 13 e mail info stemcellgb com In North America Tel 1 604 877 0713 Fax 1 604 877 0704 Toll Free Tel 1 800 667 0322 Toll Free Fax 1 800 567 2899 e mail info stemcell com www stemcell com In Europe Tel 33 0 4 76 04 75 30 Fax 33 0 4 76 18 99 63 e mail info stemcellfrance com FOR RESEARCH USE ONLY 28863 we Printed on recycled paper
6. ail when mixed with your own mouse IgG monoclonal antibody This selection cocktail along with the EasySep Magnetic Nanoparticles and EasySep Magnet or RoboSep can then be used to positively select cells with the cell surface antigen recognized by your mouse IgG monoclonal antibody See EasySep Labeling on reverse Note EasySep Do It Yourself Selection Kit is not recommended for selection of mouse cells An appropriate species specific Fc blocking antibody not supplied may be required to achieve desired purities by minimizing nonspecific binding to monocytes and macrophages PART A COCKTAIL ASSEMBLY PROCEDURE Note Components must be added in the proper order Add 15 ug of your mouse IgG monoclonal antibody dissolved in your buffer of choice to a 1 5 mL il ale Pick up the magnet and in one continuous motion invert the magnet and tube pouring off fraction The magnetically labeled cells will remain inside the tube held by the magnetic polypropylene microcentrifuge tube Record the volume The total volume of antibody must n exceed 800 uL i e antibody concentration must be at least 19 g mL Add 100 pL of component A to the vial Mix well Add 100 pL of component B to the vial Mix well Tightly cap the vial and place it into a 37 C incubator or water bath for five hours or overnight saline ot Bring the vial to a final volume of 1 0 mL by adding the appropriate volume of sterile phospha
7. nd using fluorochrome conjugated antibody clones that are not blocked by the antibody clone used in the TAC If this is not possible one of the following methods can also be used to assess purity 1 Add fluorochrome labeled antibodies at the same time as the cocktail Add the fluorochrome conjugated antibody at a concentration of 0 15 0 4 ug mL immediately after adding the cocktail to provide a strong detection signal without affecting separation performance This method labels the positive cells in the entire sample 2 Use a secondary fluorochrome conjugated antibody such as FITC labeled sheep anti mouse IgG 3 Use alternate markers if applicable COMPONENT DESCRIPTIONS EasySep Do It Yourself Component A code 18090 Contains a mouse monoclonal antibody directed against dextran This antibody has been purified from hybridoma culture supernatant by affinity chromatography using Protein A or Protein G Sepharose The mouse monoclonal antibody subclass is IgG Supplied in phosphate buffered saline EasySep Do It Yourself Component B code 18091 Contains a rat monoclonal antibody directed against the Fc region of Mouse IgGi This antibody has been purified from hybridoma culture supernatant by affinity chromatography using Protein A or Protein G Sepharose Supplied in phosphate buffered saline EasySep Magnetic Nanoparticles code 18150 A suspension of magnetic dextran iron particles in water RoboSep Vial for
8. te buffered This cocktail is now ready for use in the manual EasySep sytem If using RoboSep transfer the cocktail to the empty vial provided The cocktail is stable for at least 1 year at 4 C Do not freeze the cocktail PART B CELL SELECTION PROCEDURE This procedure is used for processing up to 5 x 10 cells per separation Some modification may be necessary for optimal results see Notes and Tips on reverse side Prepare nucleated cell suspension at a concentration of 1 x 10 nucleated cells mL in recommended medium see Notes and Tips For rare cells start with a concentration of 2 x 10 cells mL see Notes and Tips Optimizi Purity Cells must be in a 12 x 75 mm polystyrene tube in order to properly fit into the EasySep Magn Do not exceed a volume of 2 5 mL per tube For samples containing 1 x 107 cells or fewer resuspe in 100 pL ng et nd Falcon 5 mL Polystyrene Round Bottom Tubes Becton Dickinson Catalog 352058 are recommended Note If using RoboSep species specific blocker should be added to the cells before they are loaded into the carousel See below for details Add species specific FcR blocking antibody if required at a final concentration of 0 5 to 3 0 pg mL al mix well Cells e g for 2 mL of cells add 200 uL of cocktail Mix well and incubate at room temperature 15 minutes Mix EasySep Magnetic Nanoparticles to ensure that they are in a uniform suspension by pipetting or
9. th a mouse IgG monoclonal antibody Cells that express the surface antigen that is recognized by the mouse IgG monoclonal antibody are then targeted for magnetic separation using the EasySep Magnetic Nanoparticles and EasySep Magnet EASYSEP LABELING OF CELLS Target cells are specifically labeled with dextran coated magnetic nanoparticles using bispecific Tetrameric Antibody Complexes TAC These complexes recognize both dextran and the target cell surface antigen recognized by your own mouse IgG antibody Figure 1 The small size of the magnetic dextran iron particles allows for efficient binding to the TAC labeled cells and does not interfere with subsequent FACS analysis Magnetically labeled cells are then separated from unlabeled cells using the EasySep procedure reverse side Figure 1 Schematic Drawing of EasySep Magnetic Labeling of Cells Your own mouse IgG antibody is EasySep shown in red Magnetic Nanoparticle NOTES AND TIPS Preparing a Mononuclear Cell Suspension Prepare a mononuclear cell suspension from whole peripheral blood by Ficoll Paque PLUS density separation Catalog 07957 Previously frozen mononuclear cells should be incubated with 100 pg mL DNase Catalog 07900 in buffer without EDTA for at least 15 minutes at room temperature prior to labeling and separation to reduce clumping Filter clumpy suspensions through a 70 um mesh nylon strainer for optimal results If desired
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