Introduction

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

Principle of Isolation

Add FCS and a mixture of biotinylated monoclonal antibodies (Antibody Mix) against the non-NK cells to the starting sample. Add Depletion MyOne™ SA Dynabeads® to bind the non- NK cells during a short incubation. Separate the beadbound cells with a magnet. Discard the bead-bound cells and use the remaining untouched NK cells for any application.


Downstream Applications

Isolated NK cells can be used in any application, e.g.: Measuring cytokine production (immune reactions), cancer studies, functional assays, molecular studies and Flow cytometry. (for recommended products and protocols visit Immunology Research Guide.  Negatively isolated NK cells have been shown to retain cytotoxic activity against K562 cells (1,3,4).


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 Foetal Bovine Serum (FBS)/ Foetal Calf Serum (FCS).
  • Lymphoprep® for PBMC preparation (Axis Shield PoC, Norway).
  • Keep the buffers cold!


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 vigorously the recommended times to prevent trapping of CD8 T cells in the bead-bound cell fraction. Try to avoid air bubbles during pipetting.
  • This product should not be used with Dynal® MPC™-1 (cat.no. 120.01D).
  • Follow the recommended volumes and incubation times.

Protocol

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


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 NK  cell isolation

Working volume per 5 x 107 PBMC Working volume per 5 x 108 PBMC
Tube size 15 ml 50 ml
Cell volume (step 1) 500 μl 5 ml
FBS/FCS (step 2) 100 μl 1 ml
Antibody Mix (step 3) 100 μl 1 ml
Washing (step 5) 10 ml 40 ml
Resuspension (step 6) 500 μl 5 ml
Depletion MyOne Dynabeads® (step 7) 500 μl 5 ml
Volume added before
magnetic separation (step 10)*
5 ml 20 ml


* 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 300 × 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 CD8+ T 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.


Technical Recommendations

Sample Preparation

Preparation of PBMC from buffy coat to obtain low platelet numbers:

  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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General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Description of Materials

Depletion MyOne™ Dynabeads® (20 mg/ml) are uniform, superparamagnetic polymer beads (1.0 μm diameter) supplied in phosphate buffered saline (PBS), pH 7.4, containing 0.1 % bovine serum albumin (BSA) and 0.02 % sodium azide (NaN3). The Dynabeads® are coated with a monoclonal human IgG4 anti-mouse IgG antibody, which recognizes all mouse IgG subclasses and is Fc-specific. The source of the human monoclonal antibody is free of Human Immunodeficiency Virus (HIV), Hepatitis-B Virus (HBV) and Hepatitis-C Virus (HCV). The Antibody Mix contains mouse IgG antibodies for CD8, CD14, CD16 (specific for CD16a and CD16b), CD19, CD36, CD56, CDw123 and CD235a (Glycophorin A). Supplied in PBS with 0.5% BSA and 0.02% sodium azide (NaN3).

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Warnings And Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.

Avoid pipetting by mouth!

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .
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References

  1. Andersen MH et al (2005) Spontaneous Immunity against Bcl-xL in Cancer Patients. J. Immunol. 175: 2709-2714.

  2. Chemnitz JM et al (2004) SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation but only receptor ligation prevents T cell activation. J. Immunol. 173: 945-954.

  3. Jarnak-Jankovic S et al (2005) Evaluation of dendritic cells loaded with apoptotic cancer cells or expressing tumour mRNA as potential cancer vaccines against leukemia. BMC Cancer. 5:20 doi:10.1186/1471-2407-5-20.

  4. Monteiro M et al (2007) Cartography of gene expression in CD8 single cells: novel CCR7- subsets suggest differentiation independent of CD45RA expression. Blood. 109:2863-2870.

  5. van Rhee F et al (2005) NY-ESO-1 is highly expressed in poor prognosis multiple myeloma and induces spontaneous humoral and cellular immune responses. Blood. (Pre-published online): 10.1182/blood-2004-09-3707.

  6. Zioza A et al (2003) CD8+ T cells that express CD4 on their surface (CD4dim-CD8bright T cells) recognize an antigenspecific target, are detected in vivo and can be productively infected by T-tropic HIV. Blood 102: 2156-2164.

 

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113.49D.indd    Rev 001     5-May-2008