Isolation of Untouched Human CD8+ T Cells from Peripheral Blood Mononuclear Cells (PBMC)

This protocol is intended for isolation of untouched human CD8⁺ T cells from peripheral blood mononuclear cells (PBMC) by depleting B cells, NK cells, monocytes, dendritic cells, CD4⁺ T cells, granulocytes. Isolated CD8⁺ T cells are bead- and antibody-free and are suitable for any downstream application.

Principle of Isolation

A mixture of biotinylated monoclonal antibodies against the non-CD8⁺ T cells is added to the starting sample and allowed to bind to the cells. Depletion MyOne™ SA Dynabeads® are added and will bind to the antibody-labelled cells during a short incubation. The bead-bound cells are subsequently separated on a magnet and discarded. The remaining, untouched human CD8⁺ T cells can be used for any application.


Downstream Applications

Isolated CD8⁺ T cells can be used in any application, e.g.:

• Studies on CD8⁺ T cell proliferation, apoptosis and induction of anergy.
• Studies on antigen specific T cells.
• Studies on regulation of CD8⁺ T cell cytokine expression.
• Flow cytometry/FACS sorting.

Additional Materials Required

• Mixer allowing both tilting and rotation.
• Magnets: See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps.
• Isolation Buffer: Ca²⁺ and Mg²⁺ free phosphate buffered saline (PBS) (e.g. Gibco cat.no. 10010-023) 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:

• 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

• 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.
• It is important to pipette roughly the recommended times to prevent trapping of NK cells in the bead-bound cell fraction (chapter 3, step 9 and 14). Try to avoid air bubbles during pipetting.
• Follow the recommended volumes and incubation times. (Never use less than recommended volume of Dynabeads®).
• Keep the buffers cold!

Dynabeads® Washing Procedure

Dynabeads® should be washed before use.

1. Resuspend the Dynabeads® in the vial. (i.e.vortex for > 30 sec or tilt and rotate for 5 min.)


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

Preparations of PBMC

• Prepare a PBMC suspension according to the recommended sample preparation protocol (low platelet numbers).  Visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species and follow our Quicklinks for recommended sample preparation procedures.
• Resuspend the suspension at 1 x 10⁸ PBMC per ml in Isolation buffer.

Isolation Procedure

This protocol is based on 1 × 10⁷ PBMC and is scalable from 1 × 10⁷ - 5 × 10⁸ cells according to table 1.

Table 1.  Volumes for human CD8⁺ T cell isolation

* Note that the difference in volumes for small vs. large scale protocol is based on getting good working volumes during incubation with beads. A shift from small to large scale parameters may be done at any level of cells processed, as long as the resulting volume during incubation with beads is appropriate for the tubes used (more than 1/10 of total tube volume).

• The starting number of cells for this protocol is 5 x 10⁷  (see preparations above for details)

1. Transfer 500 μl (5 × 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 10 ml Isolation Buffer. Mix well by tilting the tube several times and centrifuge at 350 × 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 MyOne SA Dynabeads®.


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


9. Resuspend the bead-bound cells by vigorously pipetting >10 times using a pipette with a narrow tip opening (e.g. a 1000 μl pipette tip or a 5 ml serological pipette).


10. Add 5 ml Isolation Buffer. (When working with volumes < 1 ml during incubation with beads, add 1 ml Isolation Buffer before resuspension).


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


12. Transfer the supernatant containing the untouched human NK cells to a new tube.


13. Add 5 ml Isolation Buffer to tube containing the Dynabeads®.


14. Resuspend the bead-bound cells by vigorously pipetting as described in step 9.


15. Place the tube in the magnet for 2 min


16. Combine the two supernatants.


17. Optional: To remove residual beads, place the tube in the magnet for 2 min. and transfer cells to a new tube.

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