西亚试剂：Isolate or Deplete Human CD4+ T Cells

Dynabeads® are mixed with the cell sample in a tube. The Dynabeads® will bind to the target cells during a short incubation, and then the bead-bound cells are separated by a magnet.

Positive isolation – discard the supernatant and use the bead-bound cells for downstream molecular applications.

Depletion – discard the beadbound cells and use the remaining, untouched cells for any application.

 

实验试剂

 

Magnet (Dynal® MPC™)

Mixer allowing both tilting and rotation.

Buffer 1: PBS w/0.1% BSA and 2 mM EDTA, pH 7.4.

 

实验步骤

 

1.        Dynabeads® Washing Procedure

Dynabeads® should be washed before use.

1)        Resuspend the Dynabeads® in the vial.

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

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

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

5)        Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume of Dynabeads® (step 2).

2.        Sample Preparation

Cells can be directly isolated from any sample such as whole blood, bone marrow, MNC or tissue digests.

Please visit www.invitrogen.com/cellisolation and follow our QuickLinks for recommended sample preparation procedures.

3.        Whole Blood and Buffy Coat

Most depletions and positive isolations can use whole blood and buffy coat as a starting sample. Buffy coat is 8-10 times more concentrated than whole blood with regard to number of leucocytes. For this product you have to wash the blood/buffy coat to remove interfering soluble factors.

1)        Dilute the whole blood or buffy coat in Buffer 1 (1 2).

2)        Centrifuge at 600 x g for 10 min at 2-8°C.

3)        Discard the plasma fraction/upper layer. Resuspend blood to the original volume in Buffer 1 and buffy coat 1 1 in Buffer 1 before adding the beads.

4.        Depletion or Positive Isolation of CD4 T Cells

1)        Add the appropriate volume of Dynabeads® to the prepared sample according to table 1.

2)        Incubate for 20 min (positive isolation) or 30 min (depletion) at 2 - 8°C with gentle tilting and rotation.

3)        Place the tube in a magnet for 2 min.

4)        For depletion, transfer supernatant to a new tube for further use.

5)        For positive isolation, discard the supernatant and wash the beadbound cells 3 times by resuspending in Buffer 1 to the original sample volume, and separate using a magnet for 1 min. Never use less than 1 ml Buffer 1 in each washing step. For molecular studies, lyse cells while still attached to the beads and transfer supernatant to a new tube for protein or gene expression analysis.

 

注意事项

 

Critical Steps for Cell Isolation

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

2.        When incubating Dynabeads® and cells, the incubation temperature must be 2-8°C to reduce phagocytic activity and other metabolic processes.

3.        Never use less than 25 μl (1 x 10⁷) Dynabeads® per ml of cell sample and at least 4 Dynabeads® per target cell.

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