Isolation of Untouched Human T cells from Peripheral blood Mononuclear Cells (PBMC)

This product is intended for isolation of untouched human T cells from peripheral blood mononuclear cells (PBMC) by depleting B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes and erythrocytes. Isolated T cells are bead- and antibody-free and are suitable for any downstream application.

Principle of Isolation

A mixture of mouse IgG antibodies against the non-T cells is added to the starting sample. Depletion Dynabeads® are added and will bind to the antibody-labeled cells during a short incubation. The bead-bound cells are subsequently separated on a magnet and discarded. The remaining, untouched human T cells can be used for any application.

Downstream Applications

Isolated T cells can be analyzed in flow cytometry and used in any downstream application e.g. T cell culture, T cell activation and expansion, functional assays, molecular studies.

Sample Preparation

Untouched T cells are isolated from a PBMC suspension. We recommend using a sample preparation procedur  that gives low platelet numbers. See Technical Recommendations, or visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species for recommended sample preparation procedures.

Additional Materials Required

• Mixer allowing both tilting and rotation.
• DynaMag™ magnet: See Magnets for Molecular and Cell Separation Applications for magnet recommendations.
• Isolation buffer: Ca²⁺ and Mg²⁺ free phosphate buffered saline (PBS) (e.g. Gibco cat.no. 14190-094) supplemented with 0.1% BSA and 2mM EDTA.
• Heat inactivated Fetal Bovine Serum (FBS)/Fetal Calf Serum (FCS).
• Lymphoprep® for PBMC preparation (Axis Shield PoC, Norway).

Note:

• Keep the buffers cold.
• BSA can be replaced by human serum albumin (HSA) or 2% FBS/ FCS.
• EDTA can be replaced by 0.6% sodium citrate.
• PBS containing Ca²⁺ or Mg²⁺ is not recommended.

Critical notes

• Depletion Dynabeads® should be washed before use (see ‘Dynabeads® Washing Procedure’)
• Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads® do not settle in the tube
• This product should not be used with Dynal® MPC™-1 (cat.no. 120.01D).
• Follow the recommended volumes and incubation times.
• Avoid air bubbles during pipetting

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.


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

1. Transfer 500 μl (5 x 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.


5. Wash the cells by adding 5 ml Isolation buffer. Mix well by tilting the tube several times and centrifuge at 300 x g for 8 min at 2-8°C. Discard the supernatant.


6. Resuspend the cells in 500 μl Isolation buffer.


7. Add 500 μl pre-washed Depletion Dynabeads®.


8. Incubate for 15 min at 18-25°C with gentle tilting and rotation.


9. Resuspend the bead-bound cells by gently pipetting 5 times using a pipette with a narrow tip opening, (e.g.   1,000 μl pipette tip or a 5 ml serological pipette).


10. Add 5 ml Isolation buffer. (When working with < 1 x 10⁷ PBMC during incubation with beads, add Isolation buffer up to a total volume of 1 ml).


11. Place the tube in the magnet for 2 min.


12. Transfer the supernatant to a new tube.


13. Repeat step 10-12.


14. To remove any residual Dynabeads®, place the tube in the magnet for 2 min and transfer the supernatant to an new tube. The supernatant contains the negatively isolated human T cells.

1. Dilute 10 - 15 ml buffy coat with PBS Isolation buffer to a total volume of 35 ml at 18-25°C (RT).


2. Add the diluted buffy coat on top of 15 ml of Lymphoprep®.


3. Centrifuge at 160 x g for 20 min at RT. Allow to decelerate without brakes.


4. Remove 18-20 ml of supernatant to eliminate platelets.


5. Centrifuge at 350 x g for 20 min at RT. Allow to decelerate without brakes.


6.  Recover PBMC from the plasma/Lymphoprep® interface and transfer the cells to a 50 ml tube.


7. Wash PBMC once with Isolation buffer by centrifugation at 400 x g for 8 min at 2-8°C.


8. Wash PBMC twice with Isolation buffer by centrifugation at 225 x g for 8 min at 2-8°C and resuspend the PBMC at 1 x 10⁸ PBMC per ml in Isolation buffer.

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