Isolate or Deplete Human CD4+ T Cells - Whole Blood, Buffy Coat, MNC
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一、实验原理
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.
二、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.
三、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.
四、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.
