Isolation of Untouched Human CD4+ T Cells from Peripheral Blood Mononuclear Cells (PBMC)

Introduction

This product is intended for isolation of untouched human CD4+ T cells from peripheral blood mononuclear cells (PBMC) by depleting B cells, NK cells, monocytes, platelets, dendritic cells, CD8+ T cells, granulocytes and erythrocytes. Isolated CD4+ 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-CD4+ T cells is added to the starting sample and allowed to bind to the cells. Depletion MyOne™ 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 CD4+ T cells can be used for any application


Downstream Applications

Isolated CD4+ T cells can be used in any application, e.g.:

  • Studies on CD4+ T cell proliferation, apoptosis and induction of anergy
  • Studies on antigen specific T cells
  • Studies on regulation of CD4+ T cell cytokine expression
  • Flow cytometry/FACS sorting



Sample Preparation

Untouched CD4+ 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.
  • Magnet: We recommend DynaMag™-15(cat.no. 123.01D) for 1-15 ml samples and DynaMag™-50 (cat.no. 123.02D) for 5-50 ml samples. Dynal® MPC™ magnets may also be used, except Dynal® MPC-1 (cat.no. 120.01D).
  • 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
  • This product should not be used with Dynal® MPC™-1 (cat.no. 120.01D). See Technical Recommendations.
  • Follow the recommended volumes and incubation times.
  • Avoid air bubbles during pipetting.
  • Keep the buffers cold!

Protocol

Dynabeads® Washing Procedure

  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).

Preparations

Prepare a PBMC suspension according to the recommended sample preparation protocol (low platelet numbers, see Technical Recommendations).  Resuspend the suspension at 1 x 10 8 PBMC per ml in Isolation buffer.

Isolation Procedure

This protocol is based on 1 × 10 7 PBMC and is scalable from 1 × 10 7 - 5 × 10 8 cells according to table 1. Table 1. Volumes for human CD4 + T cell isolation (scalable from 1 × 10 7 to 5 × 10 8 PBMC). For lower cell numbers than 1 × 10 7, use the same volumes as indicated below. For higher cell numbers than 1 × 10 7, scale up the volumes accordingly.


Working volume per 1 x 107 PBMC Working volume per 5 x 108 PBMC
Cell volume (step 1)
100 μl
5 ml
FBS/FCS (step 2)
20 μl
1 ml
Antibody Mix (step 3)
20 μl
1 ml
Washing (step 5)
2 ml
Max. 40 ml
Resuspension (step 6)
100 μl
5 ml
Depletion MyOne Dynabeads® (step 7)
100 μl
5 ml
Volume added before
magnetic separation (step 10)*
1 ml
Max. 35 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 1/5 of total tube volume).

  1. Transfer 100 μl (1 × 107) PBMC in Isolation Buffer to a tube.

  2. Add 20 μl heat inactivated FCS.

  3. Add 20 μl of Antibody Mix.

  4. Mix well and incubate for 20 min at 2-8°C.

  5. Wash the cells by adding 2 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 100 μl Isolation Buffer.

  7. Add 100 μl pre-washed Depletion MyOne 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 1 ml Isolation Buffer.

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

  12. Transfer the supernatant to a new tube.

  13. Repeat step 10-12 with the tube containing the Dynabeads® and combine the two supernatants.

The supernatant contains the negatively isolated human CD4 + T cells

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.

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 (NaN 3). 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 (NaN 3).

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 .

References

  1. Aasheim HC et al (2005) Ephrin-A1 binding to CD4+ T lymphocytes stimulates migration and induces tyrosine phosphorylation of PYK2. Blood 105: 2869-2876.

  2. Jarnak-Jankovic S et all (2005) Evaluation of dendritic cells loaded with apoptotic cancer cells or expressing

  3. tumour mRNA as potential cancer vaccines against leukemia. BMC Cancer. 5:20 doi:10.1186/1471-2407-5-20.

  4. Zhang R et al (2004) CD40 ligand dysregulation in HIV infection: HIV glycoprotein 120 inhibits signalling cascades upstream of CD40 ligand transcription. J. Immunol. 172:2678-2686.

  5. Hamez-Cognasse et al (2008). Direct contact of platelets and their released products exert different effects on human dendritic cell maturation. BMC Immunology 9:54 doi:10.1186/1471-2172-9-5.

  6. Ghannam et al (2008). Human CD3 Deficiency Associated with Impairments in Dendritic Cell Differentiation, Memory B Cells, and Regulatory T Cells. The Journal of Immunology 184: 5158-5166.

113.46D_52D.indd    Rev 003     5-May-2009

For Research Use Only. Not for use in diagnostic procedures.