Purification of T cells with Robosep Kit

2015-01-13

azim58 - Purification of T cells with Robosep Kit

Original T cell Purification Protocol with Robosep
Original T Cell Purification Protocol.pdf

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

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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
  o 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:
  o 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.
  o Make sure to select which quadrants are using reagents from the
    same kit.
  o 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.
  o 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.

o 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)

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