Isolation of Untouched Human CD4+ T Cells from Peripheral Blood Mononuclear Cells (PBMC)
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实验原理
实验试剂
Mixer allowing both tilting and rotation.
Heat inactivated Fetal Bovine Serum (FBS)/Fetal Calf Serum (FCS).
Lymphoprep® for PBMC preparation.
实验步骤
1. Dynabeads® Washing Procedure
1) Resuspend the Dynabeads® in the vial.
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.
1) Transfer 100 μl (1 × 107 ) PBMC in Isolation Buffer to a tube.
2) Add 20 μl heat inactivated FCS.
3) Add 20 μl of Antibody Mix.
4) Mix well and incubate for 20 min at 2-8°C.
6) Resuspend the cells in 100 μl Isolation Buffer.
7) Add 100 μl pre-washed Depletion MyOne Dynabeads®.
8) Incubate for 15 min at 18-25°C with gentle tilting and rotation.
10) Add 1 ml Isolation Buffer.
11) Place the tube in the magnet for 2 min.
12) Transfer the supernatant to a new tube.
13) Repeat step 10-12 with the tube containing the Dynabeads® and combine the two supernatants.
The supernatant contains the negatively isolated human CD4 T cells.
注意事项
1. Dynabeads® should be washed before use (see Dynabeads® Washing Procedure).
4. Follow the recommended volumes and incubation times.
