Isolate CD4+ T Cells - Whole Blood, Buffy Coat, MNC, Bone Marrow

• Positive isolation – Dynabeads are mixed with the 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.
• Detachment – wash the positively isolated cells and then add DETACHaBEAD to gently release the cells from the beads.

• 5 ml Dynabeads CD4 4 x 10⁸ beads/ml in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN₃).
• 2 ml DETACHaBEAD CD4
• This product will process up to 2 x 10⁹ cells

• Magnet: (Dynal MPC™) - MPC-S for 20 μl to 2 ml samples, MPC-L (Cat. no. 120.20D) for 1-8 ml samples, MPC-15 (Cat. no. 120.29) for 1-15 ml samples and MPC-50 (Cat. no. 120.24) for 15-50 ml samples.
• Mixer allowing both tilting and rotation.
• Buffer 1: PBS (without Ca²⁺ and Mg²⁺ ) w/0.1% BSA and 2 mM EDTA, pH 7.4.
• Buffer 2: RPMI 1640/1% FCS (Foetal Calf Serum).

• BSA can be replaced by human serum albumin (HSA) or FCS.
• EDTA can be replaced by sodium citrate.
• PBS containing Ca²⁺ or Mg²⁺ is not recommended.

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.

Sample Preparation

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

Whole Blood and Buffy Coat

Most 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 part blood/buffy coat to 2 parts Buffer 1).


2. Centrifuge at 600 x g for 10 min at 2-8°C. Allow to decelerate slowly.


3. Discard the plasma fraction/upper layer. Resuspend blood to the original volume with Buffer 1. Buffy coat should be resuspended to double the original volume with Buffer 1.


4. Add the correct volume of beads as shown in Table 1. Please visit www.invitrogen.com for a list of recommended sample preparation procedures.

Critical Steps for Cell Isolation

Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads do not settle at the bottom of the tube. When incubating Dynabeads and cells, the incubation temperature must be 2-8°C to reduce phagocytic activity and other metabolic processes.

Table 1:   Volume of Dynabeads added per cell sample. The volumes can be scaled up as required.

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 at 2 - 8°C with gentle tilting and rotation.


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


4. Discard the supernatant and wash the bead-bound 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.


5. Resuspend the bead-bound cells in 100 μl Buffer 2 per 10⁷ MNC in the original sample, or 100 μl Buffer 2 per ml blood in the original sample. Never resuspend in less than 100 μl even if the starting sample is less than 10⁷

MNC or 1 ml blood.
Detachment

1. Add 10 μl DETACHaBEAD per 25 μl (1 x 10⁷) Dynabeads used to isolate the bead-bound cells. Never use less than 10 μl DETACHaBEAD even if less than 10⁷ Dynabeads are used. The amount of DETACHaBEAD can be directly scaled up.


2. Incubate for 45 min at room temperature with gentle mixing.


3. Place the tube in a magnet for 1 min.


4. Transfer the supernatant containing released cells to a fresh tube. To obtain residual cells, wash the beads 2-3 times in 500 μl Buffer 2 and collect the supernatant.


5. Wash detached cells thoroughly by resuspending the cells in a total volume of 10 ml Buffer 2 and centrifuge for 6 min at 400 x g to remove DETACHaBEAD.


6. Resuspend the cells in Buffer 2 or other media and use in downstream application. The isolated cells are pure, viable and are free from antibody bound to the surface.

1. Lundgren A et al. (2002) Helicobacter pylori-specific CD4⁺CD25^high regulatory T cells suppress memory T cell responses to H. pylori in infected individuals. Infection and Immunity. 71: 1755-1762.


2. Nakamura T et al. (2001) Resistance of CD4⁺ T lymphocytes to etoposideinduced apoptosis mediated by upregulation of Bc1-x L expression in patients with HTLV-1 associated myelopathy. J. Neuroimmunol. 117: 143-148.


3. Wright N et al. (2002) The chemokine stromal cell-derived factor. I alpha modulates alpha (4) beta (7) integrin-mediated lymphocyte adhesion to mucosal addressin cell adhesion molecule-1 and fibronectin. J.Immunol. 168(10):5268-5277.