Isolation of Untouched Human CD8+ T Cells from Peripheral Blood Mononuclear Cells (PBMC)
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实验原理
实验试剂
Magnets: See www.invitrogen.com/magnetselection.
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.
Resuspend the suspension at 1 x 108 PBMC per ml in Isolation buffer.
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.
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.
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.
注意事项
1. Dynabeads® should be washed before use (see Dynabeads® Washing Procedure).
