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        Isolation of Untouched Human CD8+ T Cells from Peripheral Blood Mononuclear Cells (PBMC)

        互联网

        1039

        实验原理

         

        A mixture of biotinylated monoclonal antibodies against the non-CD8 T cells is added to the starting sample and allowed to bind to the cells. Depletion MyOne™ SA Dynabeads® are added and will bind to the antibody-labelled cells during a short incubation. The bead-bound cells are subsequently separated on a magnet and discarded. The remaining, untouched human CD8 T cells can be used for any application.

        实验试剂

         

        Magnets: See www.invitrogen.com/magnetselection.

        Isolation Buffer: Ca2 and Mg2 free phosphate buffered saline (PBS) (e.g. Gibco cat.no. 10010-023) supplemented with 0.1% BSA and 2mM EDTA.

        Heat inactivated Foetal Bovine Serum (FBS)/ Foetal Calf Serum (FCS).

        Lymphoprep® for PBMC preparation.

        实验设备

         

        Mixer allowing both tilting and rotation.

        实验步骤

         

        1.        Dynabeads® Washing Procedure

        Dynabeads® should be washed before use.

        1)        Resuspend the Dynabeads® in the vial. (i.e.vortex for > 30 sec or tilt and rotate for 5 min.)

        2)        Transfer the desired volume of Dynabeads® to a tube.

        3)        Add the same volume of Isolation Buffer, or at least 1 ml, and mix.

        4)        Place the tube in a magnet for 1 min and discard the supernatant.

        5)        Remove tube from the magnet and resuspend the washed Dynabeads® in the same volume of Isolation Buffer as the initial volume of Dynabeads® (step 2)

        2.        Preparations of PBMC

        Prepare a PBMC suspension according to the recommended sample preparation protocol (low platelet numbers).  Visit www.invitrogen.com/cellisolation and follow our Quicklinks for recommended sample preperation procedures.

        Resuspend the suspension at 1 x 108 PBMC per ml in Isolation buffer.

        3.        Isolation Procedure

        1)        Transfer 500 μl (5 × 107 ) PBMC in Isolation Buffer to a tube.

        2)        Add 100 μl heat inactivated FCS.

        3)        Add 100 μl of Antibody Mix.

        4)        Mix well and incubate for 20 min at 2-8°C.

        5)        Wash the cells by adding 10 ml Isolation Buffer. Mix well by tilting the tube several times and centrifuge at 350 × g for 8 min at 2-8°C. Discard the supernatant.

        6)        Resuspend the cells in 500 μl Isolation Buffer.

        7)        Add 500 μl pre-washed Depletion MyOne SA Dynabeads®.

        8)        Incubate for 15 min at 18-25°C with gentle tilting and rotation.

        9)        Resuspend the bead-bound cells by vigorously pipetting >10 times using a pipette with a narrow tip opening (e.g. a 1000 μl pipette tip or a 5 ml serological pipette).

        10)    Add 5 ml Isolation Buffer. (When working with volumes < 1 ml during incubation with beads, add 1 ml Isolation Buffer before resuspension).

        11)    Place the tube in the magnet for 2 min.

        12)    Transfer the supernatant containing the untouched human NK cells to a new tube.

        13)    Add 5 ml Isolation Buffer to tube containing the Dynabeads®.

        14)    Resuspend the bead-bound cells by vigorously pipetting as described in step 9.

        15)    Place the tube in the magnet for 2 min

        16)    Combine the two supernatants.

        17)    Optional: To remove residual beads, place the tube in the magnet for 2 min and transfer cells to a new tube.

        注意事项

         

        1.        Dynabeads® should be washed before use (see Dynabeads® Washing Procedure).

        2.        Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads® do not settle in the tube.

        3.        It is important to pipette roughly the recommended times to prevent trapping of NK cells in the bead-bound cell fraction (chapter 3, step 9 and 14). Try to avoid air bubbles during pipetting.

        4.        Follow the recommended volumes and incubation times. (Never use less than recommended volume of Dynabeads®).

        5.        Keep the buffers cold!

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