biotechne RsDsystems MagCellect™ Mouse CD3+ T Cell Isolation KitCatalog Number: MAGM201 INTENDED USE The MagCellect Mouse CD3+ T Cell Isolation Kit is designed to isolate CD3+ T cells via a negative selection principle. The resulting cell preparation is highly enriched with CD3+ T cells. Typical purity of recovered CD3+ T cells ranges from 90-98%. BACKGROUND R&D Systems® MagCellect products are designed for the isolation of cells in a "liquid phase". R&D Systems MagCellect technology is based on the use of ferrofluids or magnetic nanoparticles that have no magnetic memory (superparamagnetic), and behave like colloidal particles. This feature allows the ferrofluids to remain in solution without the need for mixing and additionally allows for efficient diffusion kinetics during the binding reaction. The proprietary manufacturing technology of MagCellect Ferrofluids generates particles with higher ligand binding capacity per mass compared to many other larger diameter magnetic particles. PRINCIPLE OF SELECTION A mononuclear cell suspension is first incubated with the MagCellect Mouse CD3+ T Cell Biotinylated Antibody Cocktail which targets the unwanted cells. MagCellect Streptavidin Ferrofluid is next added to the reaction and the streptavidin coated nanoparticles interact with biotinylated antibody tagged cells. The tube containing the cell suspension is then placed within a magnetic field. Magnetically tagged cells will migrate toward the magnet (unwanted cell fraction), leaving the untagged cells or desired cell population in suspension to be harvested by aspiration while the tube remains in the magnetic field. The enriched cell preparation is then available for a variety of applications including tissue culture, immune status monitoring and flow cytometry. MATERIALS PROVIDED & STORAGE CONDITIONS Store the unopened kit at 2-8 °C. DO NOT FREEZE. This kit contains sufficient reagents to process 1 x 109 total cells. * Provided this is within the expiration date of the kit. OTHER SUPPLIES REQUIRED • MagCellect Magnet (R&D Systems, Catalog # MAG997) • 12 x 75 mm (5 mL) or 17 x 100 mm (15 mL) polystyrene round bottom tubes • Sterile Pasteur pipettes or transfer pipettes (ThermoFisher, Catalog # 13-711-9B or equivalent) • Sterile distilled or deionized water PRECAUTION Some components of this kit contain sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal. Global bio-techne.com [email protected] TEL +1 612 379 2956 USA TEL 800 343 7475 Canada TEL 855 668 8722 Europe | Middle East | Africa TEL +44 (0)1235 529449 China TEL +86 (21) 52380373 For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners.

CELL PREPARATION 1. Gently tease apart the mouse spleen(s) in order to generate a single cell suspension in Hanks’ Balanced Salt Solution (HBSS) (or other preferred media) supplemented with 10% bovine serum. To remove cell clumps and/or debris pass the suspended cells through a 40-70 µm nylon cell strainer. 2. Wash the cells once by filling a 15 or 50 mL centrifuge tube with HBSS + 10% serum and spinning the cells for 10 minutes at 200 x g (use a 50 mL tube when processing more than 2 spleens). 3. Decant the supernatant, disrupt the cell pellet by “racking” the tube, resuspend the cells in R&D...

TECHNICAL HINTS • If sterile cells are required following the cell selection, the entire procedure should be carried out in a laminar flow hood to maintain sterile conditions. Use sterile equipment when pipetting reagents that will be reused at a later date. • Avoid antibody capping on cell surfaces and non-specific cell tagging by working fast, keeping cells and solutions cold through the use of pre-cooled solutions and by adhering to the incubation times and temperatures specified in the protocol. Increased temperature and prolonged incubation times may lead to non-specific cell labeling thus...