COMPONENT NAME COMPONENT # QUANTITY STORAGE SHELF LIFE FORMAT RosetteSep Human Progenitor Cell Basic Pre-Enrichment Cocktail

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1 This document is available at Positive Selection Catalog #7897 EasySep Human Cord Blood CD Positive Selection For processing 000 ml of cord blood Description Isolate highly purified CD+ cells from fresh whole umbilical cord blood using a simple, two-step procedure. Fast and easy-to-use Up to 96% purity No columns required Can be combined with SepMate for consistent, high-throughput sample processing First, hematopoietic progenitor cells are pre-enriched using RosetteSep Human Progenitor Cell Basic Pre-Enrichment Cocktail (226C) with antibodies recognizing T cell, B cell, and myeloid cell surface markers. CD+ cells are then selected using EasySep Human CD Positive Selection Cocktail (8096C), which contains an antibody recognizing CD. RosetteSep binds unwanted cells to red blood cells (RBCs), forming immunorosettes, which sediment during density gradient centrifugation. The pre-enriched fraction containing CD+ cells is harvested from the interface between the plasma and density gradient medium. The pre-enriched CD+ cells are then labeled with antibodies and magnetic particles, and separated without columns using an EasySep magnet. Unwanted cells are simply poured off, while desired cells remain in the tube. Isolated CD+ cells are immediately available for downstream applications. If isolating CD+ cells from fresh cord blood samples where platelet removal is desired, we recommend using EasySep Human Cord Blood CD Positive Selection Kit II (Catalog #7896). If isolating CD+ cells from fresh adult peripheral blood or buffy coat, we recommend using Complete Kit for Human Whole Blood CD+ Cells (Catalog #086). If isolating CD+ cells from other samples, including fresh or previously frozen mobilized peripheral blood or bone marrow mononuclear cells, or from previously frozen cord blood mononuclear cells, we recommend using EasySep Human CD Positive Selection Kit (Catalog #806). Component Descriptions COMPONENT NAME COMPONENT # QUANTITY STORAGE SHELF LIFE FORMAT RosetteSep Human Progenitor Cell Basic Pre-Enrichment Cocktail EasySep Human CD Positive Selection Cocktail EasySep Dextran RapidSpheres C 8096C 2 x 2. ml 2 x ml 000 x ml Store at 2-8 C. Do not freeze. Store at 2-8 C. Do not freeze. Store at 2-8 C. Do not freeze. PBS - phosphate-buffered saline Components may be shipped at room temperature ( - 2 C) but should be stored as indicated above. Sample Preparation For available fresh samples, see CORD BLOOD Collect cord blood in a blood collection container with anticoagulant. Recommended Medium Stable until expiry date (EXP) on label. Stable until expiry date (EXP) on label. Stable until expiry date (EXP) on label. A combination of monoclonal antibodies in PBS. A combination of monoclonal antibodies in PBS. Includes an Fc receptor blocking antibody. A suspension of magnetic particles in water. EasySep Buffer (Catalog #20), RoboSep Buffer (Catalog #200), or PBS containing 2% fetal bovine serum (FBS) and mm EDTA. Medium should be free of Ca++ and Mg++. Density Gradient Medium Lymphoprep (Catalog #0780). Page of 6

2 Easy Sep Human Cord Blood CD Positive Selection Directions for Use RosetteSep Protocol See page for Sample Preparation and Recommended Medium. Refer to Table for detailed instructions regarding the RosetteSep procedure. For more rapid RosetteSep processing this product can be combined with the SepMate RUO (Catalog #860) or SepMate IVD* (Catalog #80) cell isolation tube. For more information on SepMate see the associated Product Information Sheets. * SepMate IVD is only available in select regions where it is registered as an In Vitro Diagnostic (IVD) device for the isolation of mononuclear cells (MNCs) from whole blood or bone marrow by density gradient centrifugation. Ensure that cord blood sample, recommended medium, density gradient medium, and centrifuge are all at room temperature ( - 2 C). Table. RosetteSep Human Cord Blood CD Pre-Enrichment Protocol ROSETTESEP STEP INSTRUCTIONS Standard 0 ml Tube SepMate -0 Collect cord blood sample within the volume range. - ml - 7 ml 2 Add RosetteSep Cocktail to sample. μl/ml of sample μl/ml of sample Mix and incubate. RT for 0 minutes RT for 0 minutes Dilute sample with recommended medium and mix gently. Equal volume to sample Equal volume to sample Add density gradient medium to required tube. ml ml Add diluted sample to the tube containing the density gradient medium. Layer diluted sample on density gradient medium, being careful to minimize mixing Pour or pipette diluted sample into tube 6 Centrifuge. 200 x g for 20 minutes brake off 200 x g for 0 minutes brake on 7 Collect pre-enriched cells. For platelet removal see footnote below. Harvest enriched cell layer with a pipette and transfer to new 0 ml tube** Pour supernatant into a new standard 0 ml tube 8 Wash pre-enriched cells. Top up with recommended medium Top up with recommended medium 9 Centrifuge. *** For platelet reduction see footnote below. 0 Resuspend pre-enriched cells as indicated, in recommended medium. NOTE: If working with a sample which contains a large volume of RBCs, the RosetteSep preenriched cell pellet may be slightly larger than the recommended resuspension volume. Do not add any additional recommended medium to the sample. For an original cord blood volume of: < 00 ml resuspend in 0. ml 00-0 ml resuspend in 0.7 ml > 0 ml resuspend in ml For an original cord blood volume of: < 00 ml resuspend in 0. ml 00-0 ml resuspend in 0.7 ml > 0 ml resuspend in ml The pre-enriched cells are ready for use. Continue with the EasySep or RoboSep Human Cord Blood CD Positive Selection protocol Continue with the EasySep or RoboSep Human Cord Blood CD Positive Selection protocol RT - room temperature ( - 2 C) To minimize platelet contamination, remove and discard the top third of the plasma layer before collecting the cells at the density gradient medium : plasma interface. ** Sometimes it is difficult to see the cells at the interface. For maximum recovery, remove some of the density gradient medium along with the pre-enriched cells. *** For additional platelet removal, centrifuge cells at 20 x g for 0 minutes with the. Carefully aspirate and discard the supernatant. Repeat if desired. Continue with step 0. Cell pellets from separate cord bloods, resuspended according to Table, step 0, may be combined to obtain a maximum sample volume of ml when using The Big Easy or the EasyEights EasySep magnet. Page 2 of 6

3 Easy Sep Human Cord Blood CD Positive Selection Directions for Use Manual EasySep Protocols See page for Sample Preparation and Recommended Medium. Refer to Tables 2 and for detailed instructions regarding the EasySep procedure for each magnet. Table 2. Easy Sep Human Cord Blood CD Positive Selection Protocol EASYSEP MAGNETS STEP INSTRUCTIONS EasySep (Catalog #8000) The Big Easy (Catalog #800) Prepare RosetteSep pre-enriched sample according to Table. Add sample to required tube. 0. ml 0. - ml ml (2 x 7 mm) polystyrene round-bottom tube (e.g. Corning Catalog #208) ml (7 x 00 mm) polystyrene round-bottom tube (e.g. Corning Catalog #207) 2 Add Selection Cocktail to sample. 00 μl/ml of sample 00 μl/ml of sample Mix and incubate. RT for 0 minutes RT for 0 minutes Vortex RapidSpheres. NOTE: Particles should appear evenly dispersed. 0 seconds 0 seconds Add RapidSpheres to sample. 0 μl/ml of sample 0 μl/ml of sample Mix and incubate. RT for minute RT for minutes Add recommended medium to top up the sample to the indicated volume. Mix by gently pipetting up and down 2 times. Top up to 2. ml Top up to ml for samples ml Top up to 0 ml for samples > ml Place the tube (without lid) into the magnet and incubate. RT for minutes RT for minutes 6 Pick up the magnet, and in one continuous motion invert the magnet and tube, pouring off the supernatant.* Remove the tube from the magnet; this tube contains the isolated cells. 7 Repeat steps as indicated. Steps and 6, three more times (total of x -minute separations) Steps and 6, three more times (total of x -minute separations) 8 Remove the tube from the magnet and top up the sample with recommended medium. Centrifuge. 9 Resuspend cells in desired medium. Isolated cells are ready for use Isolated cells are ready for use RT - room temperature ( - 2 C) Cell pellets from separate cord bloods, resuspended according to Table, step 0, may be combined to obtain a maximum sample volume of ml when using The Big Easy EasySep magnet. * Leave the magnet and tube inverted for 2 - seconds, then return upright. Do not shake or blot off any drops that may remain hanging from the mouth of the tube. Page of 6

4 Easy Sep Human Cord Blood CD Positive Selection Table. Easy Sep Human Cord Blood CD Positive Selection Protocol STEP 2 INSTRUCTIONS Prepare RosetteSep pre-enriched sample according to Table. Add sample to required tube. Add Selection Cocktail to sample. Mix and incubate. Vortex RapidSpheres. NOTE: Particles should appear evenly dispersed. Add RapidSpheres to sample. Mix and incubate. Add recommended medium to top up sample to the indicated volume. Mix by gently pipetting up and down 2 times. Place the tube (without lid) into the magnet and incubate. EASYSEP MAGNET EasyEights (Catalog #80) ml tube 0. - ml ml (7 x 00 mm) polystyrene round-bottom tube (e.g. Corning Catalog #207) 00 μl/ml of sample RT for 0 minutes 0 seconds 0 μl/ml of sample RT for minutes Top up to ml for samples ml Top up to 0 ml for samples > ml RT for 0 minutes 6 7 Carefully pipette** (do not pour) off the supernatant. Remove the tube from the magnet; this tube contains the isolated cells. Add recommended medium to top up sample to the indicated volume. Mix by gently pipetting up and down 2 - times. Place the tube (without lid) into the magnet and incubate. Top up to ml for samples ml Top up to 0 ml for samples > ml RT for minutes 8 Carefully pipette** (do not pour) off the supernatant. Remove the tube from the magnet; this tube contains the isolated cells. 9 Repeat steps as indicated. Steps 7 and 8 (total of x 0-minute and 2 x -minute separations) 0 Remove the tube from the magnet and top up the sample with recommended medium. Centrifuge. Resuspend cells in desired medium. Isolated cells are ready for use RT - room temperature ( - 2 C) Cell pellets from separate cord bloods, resuspended according to Table, step 0, may be combined to obtain a maximum sample volume of ml. ** Collect the entire supernatant, all at once, into a single pipette (e.g. for the EasyEights ml tube use a 0 ml serological pipette). Page of 6

5 Easy Sep Human Cord Blood CD Positive Selection Directions for Use Fully Automated RoboSep Protocol See page for Sample Preparation and Recommended Medium. Refer to Table for detailed instructions regarding the RoboSep procedure. Table. RoboSep Human Cord Blood CD Positive Selection Protocol STEP INSTRUCTIONS RoboSep (Catalog #20000 and #2000) Prepare RosetteSep pre-enriched sample according to Table. Add sample to required tube ml ml (7 x 00 mm) polystyrene round-bottom tube (e.g. Corning Catalog #207) 2 Select protocol. Human CD Positive Selection III from CB 7897 Human CD Positive Selection III from CB high purity Vortex RapidSpheres. NOTE: Particles should appear evenly dispersed. Load the carousel. Start the protocol. Unload the carousel when the run is complete and remove the tube from the magnet. Centrifuge. 0 seconds Follow on-screen prompts Press the green Run button 6 Resuspend cells in desired medium. Isolated cells are ready for use Cell pellets from separate cord bloods, resuspended according to Table, step 0, may be combined to obtain a maximum sample volume of ml. Page of 6

6 Easy Sep Human Cord Blood CD Positive Selection Notes and Tips ASSESSING PURITY The EasySep Human Cord Blood CD Positive Selection Cocktail uses a class II anti-cd antibody clone that may block some class I and II anti-cd antibody clones used to assess purity by flow cytometry. For purity assessment by flow cytometry use one of the following class III fluorochrome-conjugated anti CD antibody clones and a fluorochrome-conjugated anti-cd antibody: Anti-Human CD Antibody, Clone 8 (Catalog #600), Anti-Human CD Antibody, Clone 8G2 (Catalog #6002), clone AC6, or clone BirmaK, and Anti-Human CD Antibody, Clone HI0 (Catalog #6008) Isolated CD+ cells can be expanded and/or differentiated into mature hematopoietic cells of specific lineages using StemSpan Serum-Free Expansion Media and Supplements (for more information, visit The frequency of erythroid (BFU-E/CFU-E), myeloid (CFU-GM), and multilineage (CFU-GEMM) progenitor cells can be assessed in colony-forming unit (CFU) assays in semi-solid culture media using MethoCult H0 Optimum (Catalog #00) or MethoCult H0 Optimum Without EPO (Catalog #00). Data Starting with fresh cord blood, the CD+ cell content of the isolated fraction is typically 87 ± 2% (mean ± SD, n = 0; data obtained using the purple EasySep Magnet). The CD+ cell content of the starting sample is typically 0. ± 0.2% (mean ± SD). STEMCELL TECHNOLOGIES INC. S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 8. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. Copyright 206 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, EasyEights, EasySep, MethoCult, RapidSpheres, RoboSep, RosetteSep, SepMate, and StemSpan are trademarks of STEMCELL Technologies Inc. Lymphoprep is a trademark of AXIS-SHIELD. All other trademarks are the property of their respective holders. While STEMCELL has made all reasonable efforts to ensure that the information provided by STEMCELL and its suppliers is correct, it makes no warranties or representations as to the accuracy or completeness of such information. Page 6 of 6