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