APC Selection Kit
Contents
1. EasySep Procedure 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 PlS pdf for additional instructions If using RoboSep The Fully Automated Cell Separator please follow Step 1 then select the appropriate program or refer to the RoboSep user manual Add EasySep APC selection cocktail to single cell suspension labeled with APC conjugated antibody EasySep Incubate 15 minutes Add EasySep magnetic nanoparticles EasySep Incubate 10 minutes Place tube in magnet for 5 minutes Pour off supernatant Positively selected cells remain in tube After two rinses collect cells simply by removing tube ays Positive Selection 7EasySep APC Selection Kit CATALOG 18451 18452 18453 And More Version 1 1 0 Some modifications to this protocol may be necessary for optimal results See Notes and Tips on reverse side Note If using RoboSep APC conjugated antibody must be transferred to the empty vial provided before loading into the carousel i This procedure is used for processing up to 5 x 10 cells per separation Prepare 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 cell concentration of 2 x 10 cells mL see Notes and Tips Op2. cells e g for 2 mL of cells add 100 uL of nanoparticles Mix well and incubate at room temperature for 10 minutes Bring the cell suspension to a total volume of 2 5 mL by adding recommended medium Mix the cells in the tube by gently pipetting up and down 2 3 times Place the tube without cap into the magnet Set aside for 5 minutes Pick up the magnet and in one continuous motion invert the magnet and tube pouring off the supernatant fraction The magnetically labeled cells will remain inside the tube held by the magnetic field of the EasySep Magnet Leave the magnet and tube in inverted position for 2 3 seconds then return to 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 recommended medium Mix the cell suspension by gently pipetting up and down 2 3 times Place the tube back in the magnet and set aside for 5 minutes Repeat Steps 7 and 8 and then Step 7 once more for a total of three 5 minute separations in the magnet For mouse separations or low frequency cell types additional separations may improve purity see Notes and Tips Optimizing Purity Remove tube from magnet and resuspend cells in an appropriate amount of desired medium The positively selected cells are now ready for use Users of this selection kit should ensure that they are entitled to use the antibody of interest StemCell Technologies is not
3. recycled paper
4. responsible for patent infringements or violations that may occur when using this product StemCell Technologies In the United Kingdom In Europe Tel 44 0 20 7691 3561 Tel 33 0 4 76 04 75 30 Fax 33 4 76 18 99 63 Fax 33 0 4 76 18 99 63 Toll Free within United Kingdom e mail info stemcellfrance com 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 March 2007 29078 FOR RESEARCH USE ONLY te Printed on recycled paper Catalog 18451 Human Catalog 18452 Mouse Catalog 18453 Other Components e EasySep APC Selection Cocktail For labeling 10 total cells For labeling 10 total cells For labeling 10 total cells 1 0 mL e Species Specific Blocker Human amp Mouse Only Anti Human CD32 Fcy RII Blocker 18451 Only Mouse FcR Blocker 18452 Only e EasySep Magnetic Nanoparticles e RoboSep Vial for Primary Conjugated Antibody not required for manual use 1 0 mL 0 1 mL 1 0 mL 1 vial REQUIRED EQUIPMENT Easysep Magnet Catalog 18000 or The Big Easy EasySep Magnet Catalog 18001 or RoboSep Catalog 20000 PRODUCT DESCRIPTION AND APPLICATIONS Easysep APC Selection Cocktail and EasySep Magnetic Nanoparticles target cells that are labeled with APC
5. 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 Mouse spleen and bone marrow cells should be prepared following standard procedures and filtered through a 70 um mesh nylon strainer Recommended Medium The recommended medium is PBS with 2 FBS Catalog 07905 and 1 mM EDTA Medium should be Ca and Mg free Assessing Purity Since the positively selected cells have already been labeled by the APC conjugated antibody their purity can be assessed by flow cytometry without additional labeling 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 7 and 8 an additional 1 3 times Please note that recovery will decrease with each additional round of separation When isolating cells representing less than 2 of the initial population the purity of the enriched sample may be improved by starting with a cell concentration of 2 x 10 cells mL For some cell types decreasing the amount of APC Selection Cocktail added can increase purity while decreasing recovery This will also reduce side scatter during subsequent flow cytometry analysis Performance may also be improved by adding a wash step After incubating the sample with APC conjugated antibody Step 3 wash once with 10 fold excess medium and resuspend to or
6. al antibodies purified from hybridoma culture supernatant by affinity chromatography using Protein A or Protein G Sepharose These antibodies are bound in bispecific tetrameric antibody complexes TAC which are directed against APC allophycocyanin and dextran These mouse monoclonal antibodies are of subclass IgG This cocktail is supplied in phosphate buffered saline It should be noted that this product is a biological reagent and as such cannot be completely characterized or quantified Some variability is unavoidable Anti Human CD32 Fcy Ril Blocker code 18520 This antibody recognizes human CD32 Fcy Ril present on monocytes platelets macrophages and granulocytes Supplied in phosphate buffered saline EasySep Mouse FcR Blocker code 18720 This antibody recognizes mouse CD16 CD32 Fcy III II Receptor present on monocytes macrophages and other FcR cells Supplied in phosphate buffered saline in 0 1 BSA and 0 1 sodium azide 100 101 102 103 104 CD4 APC EasySep Magnetic Nanoparticles code 18150 A suspension of magnetic dextran iron particles in water RoboSep Vial for Primary Conjugated Antibody code 18550 Empty vial compatible with RoboSep the fully automated cell separator If using RoboSep transfer APC conjugated antibody to 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 APC Selection C
7. allophycocyanin conjugated antibodies not supplied for magnetic separation The species specific FcR blocker anti CD32 Human anti CD16 32 Mouse is used to prevent non specific selection of monocytes and macrophages Note When selecting cells from other species an appropriate species specific FcR blocking antibody may be required to achieve desired purities A final concentration of 0 5 3 0 g mL is recommended for the blocking antibody These reagents are designed to positively select cells labeled with APC conjugated antibody EASYSEP LABELING OF CELLS Target cells that have been specifically labeled with APC conjugated antibody are then labeled with dextran coated magnetic nanoparticles using bispecific Tetrameric Antibody Complexes TAC These complexes recognize both dextran and the APC molecule on the APC conjugated antibody Figure 1 Magnetically labeled cells are then separated from unlabeled cells using the EasySep procedure reverse side EN s ur va gt w TAN eee Figure 1 Calli SaN a We Schematic Drawing of EasySep vn s d TAC Magnetic Labeling of Cells S IP _ a b i a Nance 9 OX NOTES ANDTIPS Preparing a Cell Suspension Prepare a mononuclear cell suspension from human peripheral blood by Ficoll Paque PLUS density separation Catalog 07957 Previously frozen mononuclear cells should be incubated with 100 g mL DNase Catalog 07900 in medium without EDTA for at least
8. iginal volume Please note that this wash step can not be automated by RoboSep Optimizing Recovery Recovery of positively selected APC labeled cells is dependent on the quality of the APC conjugated antibody used 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 It is important to add sufficient APC conjugated antibody to ensure a significant fluorescence intensity of the target cells as there is a strong correlation between fluorescence intensity and cell recovery We recommend that the fluorescence intensity of the positively selected cells be at least 100 fold 2 logarithms greater than that of the negative control for adequate recovery Recovery may also be improved by increasing separation times in the magnet from 5 minutes to 10 minutes for each round of separation EasySep j And More Positive Selection sh Product Information Sheet EXAMPLE EASYSEP APC SELECTION PROFILE HUMAN CD4 Start 27 7 CD4 Cells Selected 95 5 CD4 Cells 300 300 Counts Counts 0 0 i 100 101 102 103 104 100 101 102 103 104 CD4 APC CD4 APC EXAMPLE EASYSEP APC SELECTION PROFILE MOUSE CD4 Start 19 2 CD4 Cells Selected 99 4 CD4 Cells 250 250 Counts Counts 0 0 100 101 102 103 104 CD4 APC COMPONENT DESCRIPTIONS EasySep APC Selection Cocktail code 18451C This cocktail contains a combination of monoclon
9. ocktail Stable at 4 C for two years Do not freeze this product Contents sterile in unopened tube This product may be shipped at room temperature and should be refrigerated upon receipt Human or Mouse FcR Blocker Stable at 4 C for two years human or one year mouse Contents sterile in unopened tube This product may be shipped at room temperature and should be refrigerated upon receipt Do not freeze EasySep Magnetic Nanoparticles Stable at 4 C for two years Contents sterile in unopened tube This product may be shipped at room temperature and should be refrigerated upon receipt Hazardous Ingredient Sodium Azide Avoid exposure to skin and eyes ingestion and contact with heat acids and metals Wash exposed skin with soap and water Flush eyes with water Dilute with running water before discharging into plumbing 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 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 29078 te Printed on
10. timizing Purity For samples containing 10 cells or fewer resuspend in 100 uL Do not exceed an initial volume of 2 5 mL per tube Cells must be placed in a 12 x 75 mm polystyrene tube to properly fit into the EasySep Magnet Falcon 5 mL Polystyrene Round Bottom Tubes Becton Dickinson Catalog 352058 are recommended Note If using RoboSep species specific FcR blocking antibody should be added to the cells before they are loaded into the carousel See Step 2 below for details Add species specific FcR blocking antibody at 100 uL mL for human cells supplied with Catalog 18451 or 10 uL mL for mouse cells Supplied with Catalog 18452 and mix see Product Description and Applications reverse side for other species Catalog 18453 Add APC conjugated antibody at a final concentration of 0 3 3 0 ug mL Mix well and incubate at room temperature for 15 minutes Note Titrate APC conjugated antibody for optimal purity and recovery Cell recovery increases with increasing fluorescence intensity of the APC labeled cells However excess labeling antibody can reduce purity Add EasySep APC Selection Cocktail at 100 uL mL cells e g for 2 mL of cells add 200 uL of cocktail Mix well and incubate at room temperature for 15 minutes Mix EasySep Magnetic Nanoparticles to ensure that they are in a uniform suspension by vigorously pipetting up and down more than 5 times Vortexing is not recommended Add the particles at 50 uL mL
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