Human Whole Blood CD45 Depletion Kit
Contents
1. EasySep Procedure af Positive Selection EasySep muman Whole Blood Note This procedure is designed for use with The Big Easy EasySep Magnet 18001 If using RoboSep the Fully Automated Cell Separator please select the appropriate program or refer to the RoboSep user manual CD45 Depletion Kit gt zim Add EasySep depletion cocktail CATALOG 18289 Version 1 0 0 EasySep 1 This procedure is used for processing up to 85 mL of whole blood per separation Collect whole blood in a heparinized blood collection tube or bag Add HetaSep at a ratio of 1 part HetaSep to 5 parts whole blood For large samples Falcon 50 mL polypropylene tubes Becton Dickinson Catalog 352070 are recommended For smaller samples Falcon 14 mL polystyrene tubes Becton Dickinson Catalog 352057 are recommended Please note that this procedure will give optimal results when tubes are at least HetaSep treated whole blood 80 full Nucleated cell recovery can decrease when using tubes less than half full see Protocol Step 1 for due to shorter sedimentation distance sample preparation details Mix well and centrifuge at 50 x g for 5 minutes with the brake off Allow cells to sit for 5 minutes Do not allow to sit for more than 5 minutes as the leukocytes will begin to settle into the interface Incubate Please note that older blood samples will settle more slowly a 5 minute sp2. bound in bispecific Tetrameric Antibody Complexes which are directed against CD45 and dextran The mouse monoclonal antibody subclass is 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 EasySep Magnetic Nanoparticles code 18150 A suspension of magnetic dextran iron particles in water HetaSep code 07806 Hetastarch solution used to separate nucleated cells from erythrocytes in whole blood STABILITY AND STORAGE EasySep Human Whole Blood CD45 Depletion Cocktail 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 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 HetaSep Stable at 4 C for at least two years Contents sterile in unopened bottle This product may be shipped at room temperature and should be refrigerated upon receipt See Material Safety Data Sheet for more information StemCell Technologies 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 the Unit
3. centrifuge at 200 x g for 10 minutes discard supernatant resuspend in recommended medium at 1 10 of the starting volume and proceed with step 2 Recommended Medium The recommended medium is PBS containing 2 FBS Catalog 07905 and 1 mM EDTA Media should be Ca and Mg free Assessing Purity Purity of the CD45 cell fraction may be assessed by flow cytometry after staining with fluorochrome conjugated anti CD45 e g FITC anti CD45 Catalog 10417 and anti Glycophorin A e g PE anti Glycophorin A Catalog 10523 For labeling up to 100 mL whole blood 1 0 mL 1 0 mL 20 mL EasySep l D Positive Selection af Product Information Sheet TYPICAL EASYSEP CD45 DEPLETION PROFILE Start 98 3 CD45 Cells and 1 7 CAMA cells Depleted 2 log depletion of CD45 Cells seeded into whole blood and processed with HetaSep T 2 CD45 APC 102 CD45 APC 102 5E11 FITC 5E11 FITC Starting with whole blood the depletion of CD45 cells ranges from 2 4 log Plots were gated on Glycophorin A negative cells to exclude residual red blood cells t5E11 is an antibody against an epithelial cell surface antigen expressed on CAMA cells COMPONENT DESCRIPTIONS EasySep Human Whole Blood CD45 Depletion Cocktail code 18289C This cocktail contains a combination of monoclonal antibodies purified from hybridoma culture supernatant by affinity chromatography using Protein A or Protein G Sepharose These antibodies are
4. ed Kingdom Tel 44 0 20 7537 7565 Tel 33 0 4 76 04 75 30 Fax 44 0 20 7515 5408 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 Europe FOR RESEARCH USE ONLY te Printed on recycled paper
5. in at Add EasySep 15 minutes 200 x g with the brake off may be required to achieve full separation magnetic nanoparticles Remove plasma layer little or no RBCs and dilute 1 1 with recommended medium see Notes and Tips Centrifuge at 200 x g for 10 minutes Remove supernatant and resuspend cells in 1 10 of the starting volume in a 14 mL polystyrene tube Cells must be placed in a 14 mL polystyrene tube to properly fit in the EasySep magnet Do not exceed a volume of 8 5 mL Falcon 14 mL Polstyrene Round Bottom Tubes Becton Dickinson Catalog 352057 are recommended 2 Add EasySep Whole Blood Depletion Cocktail at 100 uL mL of sample mixture e g for 2 mL of sample mixture add 200 pL of cocktail Mix well and incubate at room temperature for 15 minutes 3 Mix EasySep Magnetic Nanoparticles to ensure that they are in a uniform suspension by pipetting up and down vigorously more than 5 times Vortexing is not recommended Add the particles at 100 uL mL of sample mixture e g for 2 mL of sample mixture add 200 uL of particles Mix well and incubate at room temperature for 10 minutes 4 If total volume is less than 2 5 mL add recommended medium to 5 mL otherwise add recommended medium to 10 mL Mix the cells in the tube by gently pipetting Heeb up and down 2 3 times Place the tube without cap into the magnet Set aside for minutes ten minutes 5 Pick up The Big Easy EasySep Magnet and in
6. one continuous motion invert the magnet and tube pouring off the supernatant fraction into a new tube The magnetically labeled cells will remain inside the tube held by the magnetic field of the 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 Pour off supernatant of the tube CD45 depleted cells into l a new tube 6 The cells in the supernatant fraction are depleted of CD45 expressing cells and are now ready for use Place tube in magnet for 10 minutes StemCell Technologies In North America In the United Kingdom In Europe Tel 1 604 877 0713 Tel 44 0 20 7537 7565 Tel 33 0 4 76 04 75 30 Fax 1 604 877 0704 Fax 44 0 20 7515 5408 Fax 33 0 4 76 18 99 63 Toll Free Tel 1 800 667 0322 Toll Free within United Kingdom e mail info stemcellfrance com Toll Free Fax 1 800 567 2899 Tel 0800 731 27 14 e mail info stemcell com Fax 0800 731 27 13 www stemcell com e mail info stemcellgb com August 2006 FOR RESEARCH USE ONLY te Printed on recycled paper Catalog 18289 Components EasySep Human Whole Blood CD45 Depletion Cocktail EasySep Magnetic Nanoparticles HetaSep REQUIRED EQUIPMENT The Big Easy EasySep Magnet Catalog 18001 or RoboSep Catalog 20000 PRODUCT DESCRIPTION AND APPLICATIONS EasySep Human Whole Blood CD45 De
7. pletion Cocktail and EasySep Magnetic Nanoparticles label CD45 cells for magnetic separation These reagents are designed to deplete CD45 cells cells expressing the CD45 antigen from fresh whole blood The CD45 antigen is expressed on all human leukocytes EASYSEP LABELING OF HUMAN CELLS 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 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 flow cytometric analysis Magnetically labeled cells are then separated from unlabeled cells using the EasySep procedure reverse side I amp WH v L 4 we C oe t 2 Dextran Figure 1 Cell wae lt Schematic Drawing of EasySep TAC amp k gt S d Magnetic Labeling of Human Cells i l O AO D s Complex EasySep lh od Magnetic J f Nanoparticle NOTES AND TIPS Recovery and Lysis This no lysis kit is designed for mid range recovery of CD45 cells along with between 2 and 4 log depletion of CD45 cells If higher recovery of CD45 cells is desired particles and cocktail may be titrated to achieve this If lysis of red blood cells is acceptable then add 10 parts ammonium chloride Catalog 07850 to one part whole blood Incubate for 15 minutes on ice
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