Purification+of+T+cells+with+Robosep+Kit

Original T cell Purification Protocol with Robosep

see also: Negative Selection Mouse T Cell Enrichment Kit 19751

__Original Amount of Reagents__ Normal Rat Serum: 2 mL EasySep Mouse T Cell Enrichment Cocktail: 500 uL EasySep Biotin Selection Cocktail 2: 1000 uL EasySep D Magnetic Particles: 1000 uL

__Amount of Reagents used per mL of sample__ Normal Rat Serum: 50 uL/ (mL of cells) EasySep Mouse T Cell Enrichment Cocktail: 50 uL/(mL of cells) EasySep Biotin Selection Cocktail 2: 100 uL/(mL cells) EasySep D Magnetic Particles: 75 uL/(mL cells)

Kit Components

EASYSEpTM MOUSE T CELL ENRICHMENT COCKTAIL CODE #19751 C.2 This cocktail contains a combination of biotinylated monoclonal antibodies, These antibodies are directed against cell surface antigens on mouse cells of hematopoietic origin (CDII b, CDI9, CD45R, CD49b, TERI19). This cocktail is supplied in PBS. It should be noted that this product is a biological reagent, and as such cannot be completely characterized or quantified. Some variability is unavoidable.

EASYSEpTM BIOTIN SELECTION COCKTAIL 2 CODE #19653 This cocktail is a combination of two mouse IgG, monoclonal antibodies against biotin and dextran. These antibodies are bound in bispecific Tetrameric Antibody Complexes directed against mouse IgG,. This cocktail is supplied in PBS. It should be noted that this product is a biological reagent, and as such cannot be completely characterized or quantified. Some variability is unavoidable.

EASYSEpTM D MAGNETIC PARTICLES A suspension of magnetic dextran iron particles in TRIS buffer.

NORMAL RAT SERUM CODE #19250 CODE #13551 This normal rat serum is used to prevent non-specific binding of rat antibodies to mouse cells. Serum has been certified by the manufacturer to be mycoplasma-free.

RECOMMENDED MEDIUM The recommended medium is RoboSep Buffer (Cat #20104), or phosphate buffered saline (PBS) + 2% Fetal Bovine Serum (FBS) (Cat #07905) with 1 mM EDTA added. Hanks' Balanced Salt Solution (Hanks' BSS) (Catalog #37250) can be used in place of PBS (Catalog #37350). Medium should be Ca++ and Mg++ free.

Basic Process of Kit The EasySep kits isolate cells by using tetrameric antibody complexes. There are two antibodies attached to this tetrameric complex. One antibody binds to dextran which the magnetic beads are coated with, and one antibody binds to biotin which the specific cell binding antibodies possess. These magnetic particles and things they bind will then get trapped in the magnet and other things will be transferred to the negative fraction. In the case of this specific kit, biotinylated monoclonal antibodies against CD11b, CD19, CD45R, CD49b, and TER119 are used to trap cells in the magnet and CD3+ T splenocytes (as well as other possible cells) are collected in the negative fraction.

Protocol
 * May want to place carousel in 4 C for a time before use.
 * Prepare a nucleated cell suspension
 * Disrupt spleen in recommended medium. Centrifuge at 300Xg for 10 minutes and resuspend cell pellet at about 1*10^8 cells/mL in recommended medium in a volume of 500 uL-8 mL (up to 8X10^8 cells total) in a 14 mL (17X100 mm) polystyrene tube to properly fit into the RoboSep carousel. Ammonium chloride treatment is not recommended when preparing the cells for the separation. The use of fewer than 5*10^7 cells per separation may result in sub-optimal performance.
 * Recommended Density: 1*10^8 cells/mL
 * Recommended Volume: 500 uL- 8 mL
 * Max cells: 8*10^8 cells
 * Container: 14 mL polystyrene tube
 * Add the Normal Rat Serum provided with the kit at 50 uL/mL of cells (e.g. for 2 mL of cell suspension, add 100 uL of rat serum. This rat serum is used to prevent the non-specific binding of rat antibodies to mouse cells.
 * Select the appropriate RoboSep protocol:
 * RoboSep protocols can be optimized for high T cell purity or high T cell recovery.
 * Mouse T cell Negative Selection 19751 - high purity (D particles)
 * Mouse T cell Negative Selection 19751 - high recovery (D particles)
 * Load the RoboSep carousel as directed by the on-screen prompts. Vortex the EasySep D Magnetic Particles for 30 seconds before loading. Ensure that particles are in a uniform suspension with no visible aggregates. When all desired quadrants are loaded, press the green "Run" button. All cell labeling and separation steps will be performed by RoboSep.
 * Make sure to select which quadrants are using reagents from the same kit.
 * Do not be alarmed if the Robosep machine does not seem to be processing the quadrants in any logical order.
 * When cell separation is complete, remove the tube containing the enriched cells from the RoboSep carousel. The T cells will be in the negative fraction since all of the CDII b, CDI9, CD45R, CD49b, and TERI19 cells will have been removed from the T cell solution, and these non-T cells will be found in the tube in the magnet.
 * Notes about location of tube
 * After the 2-quadrant high purity protocol, collect the enriched cells in the 14 mL tube located to the left of the magnet in the second quadrant
 * After the 1-quadrant high recovery protocol, collect the enriched cells in the 50 mL tube located to the left of the tip rack in the first quadrant.
 * The enriched cells are now ready for use.
 * Cells can be assessed for purity if desired. Purity of T cells can be measured by flow cytometry after staining with a fluorochrome-conjugated anti-CD3 (e.g. PE, anti-CD3, Cat #10800) or anti-CD90 (e.g. PE anti-CD90 Cat #10824) antibody or a combination of other T cell specific antibodies, e.g. anti-CD4 and anti-CD8 (e.g. FITC anti-CD4 Cat # 10701, and PE anti-CD8 Cat # 10803)