Introduction

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

Principle of Isolation

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

Downstream Applications

Isolated Human B cells can be used in any application, e.g.: Antigen presentation by B cells and interaction with other cells of the immune system, analysis of B cell immunoglobulin class switching and somatic hypermutation, analysis of B cell activation, proliferation and differentiation, B cell signalling pathway studies and Flow cytometry. For recommended products and protocols visit Immunology Research Guide


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.
  • Mo-DC Culture Media: RPMI-1640w/10%  Foetal 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 B cells in the bead-bound cell fraction. Try to avoid air bubbles during pipetting.
  • Follow the recommended volumes and incubation times. (Never use less than recommended volume of Dynabeads®)
  • It is important to keep cells and buffers cold when working with B cells.


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

Table 1.  Volumes for human B cell isolation

Volumes per 5 x 107 PBMC Volumes per 5 x 108 PBMC
Tube size 15 ml 50 ml
Cell volume (step 1) 500 μl 5 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 SA 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 of Antibody Mix.

  3. Mix well and incubate for 20 min at 2–8°C.

  4. 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.

  5. Resuspend the cells in 500 μl Isolation Buffer.

  6. Add 500 μl pre-washed Depletion MyOne SA Dynabeads®.

  7. Incubate for 15 min at 18–25 °C with gentle tilting and rotation.

  8. 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).

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

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

  11. Transfer the supernatant, containing the untouched human B cells, to a new tube.

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

  13. Resuspend the bead-bound cells by vigorously pipetting as described in step 8.

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

  15. Combine the two supernatants.

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


General Information

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

Description of Materials

Depletion MyOne™ SA Dynabeads® are uniform, superparamagnetic polystyrene beads (1.0 μm diameter) coated with streptavidin (SA). The Antibody Mix contains biotinylated mouse IgG antibodies for CD2, CD14, CD16 (specific for CD16a and CD16b), CD36, CD43 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 tyrosinebased 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) recognise an antigenspecific target, are detected in vivo and can be productively infected by T-tropic HIV. Blood 102: 2156-2164.

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113.51D.indd   Rev 000     5-May-2008