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        Isolation of Untouched Human B Cells by Depletion of Non-B Cells

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        731

        实验原理

         

        Add a mixture of biotinylated monoclonal antibodies (Antibody Mix) against the non-B cells to the starting sample. Add Depletion MyOne SA Dynabeads® to bind the non-B cells during a short incubation. Separate the bead-bound cells with a magnet. Discard the bead-bound cells and use the remaining, untouched human B cells for any application.

        实验试剂

         

        Mixer allowing both tilting and rotation.

        Magnets

        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.

        Mo-DC Culture Media: RPMI-1640w/10%  Foetal Calf Serum (FCS).

        Lymphoprep® for PBMC preparation

        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.        Isolation Procedure

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

        2)        Add 100 μl of Antibody Mix.

        3)        Mix well and incubate for 20 min at 2–8°C.

        4)        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.

        5)        Resuspend the cells in 500 μl Isolation Buffer.

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

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

        8)        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).

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

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

        11)    Transfer the supernatant, containing the untouched human B cells, to a new tube.

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

        13)    Resuspend the bead-bound cells by vigorously pipetting as described in step 8.

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

        15)    Combine the two supernatants.

        16)    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 B cells in the bead-bound cell fraction. Try to avoid air bubbles during pipetting.

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

        5.        It is important to keep cells and buffers cold when working with B cells.

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