Enrich Untouched Mouse Dendritic Cells

Introduction

Enrich untouched mouse Dendritic Cells (DCs) by depleting T cells, mIgM+ B cells, NK cells, erythrocytes and most granulocytes from mouse spleen or lymph node cells with this product. The DC enriched population is bead- and antibody-free and intended for further isolation of any DC subpopulation by flow sorting.

DCs in the enriched population are characterized by expression of CD11c and absence of lineage markers other than those co-plasmacytoid DCs. This kit provides high recovery of CD11c+ cells suitable for further FACS enrichment of any subpopulation, including DCs positive for CD4, CD8a, B220 and CD19 (1-4).

Principle of Isolation

Add a mixture of biotinylated monoclonal antibodies against non-DC cells to your starting sample. Add Depletion MyOne™ SA Dynabeads and allow them to bind to the non-DCs during a short incubation. Separate the beadbound cells with a magnet. Discard the bead-bound cells and use the remaining untouched, enriched cell population for further flow sorting into the DC subpopulation(s) of interest

Description of Materials

Materials Supplied

  • 5 ml Depletion MyOne SA Dynabeads  --  Depletion MyOne SA Dynabeads are uniform, superparamagnetic polystyrene beads (1.0 μm diameter) coated with streptavidin (SA). Supplied at 10 mg beads per ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide.
  • 1 ml Antibody Mix for Mouse DC kit  --  The Antibody Mix contains an optimized mixture of biotinylated monoclonal antibodies against mouse CD2, CD3ε, CD49b, mIgM and Ter-119 supplied in phosphate buffered saline (PBS), pH 7.4, containing 0.02% sodium azide.
  • The kit will process approximately 4 x 109 leucocytes, corresponding to 5 x 108 density gradient-prepared leucocytes.



Additional Materials Required

  • Magnet (Dynal MPC™): See magnet recommendations.
  • Mixer allowing both tilting and rotation.
  • Buffer 1: PBS (without Ca2+ and Mg2+) w/0.1% BSA and 2 mM EDTA, pH 7.4.
  • Buffer 2: PBS (with Ca2+ and Mg2+), and 0.1% BSA, pH 7.4
  • DNAse
  • 0.5M EDTA
  • Density gradient medium – (e.g., Nycodenz® 1.077 g/cm3 or equivalent)
  • Cell strainer, 70 μm


Important Notes:

  • It is critical to keep the buffers cold (2-8˚C) during the enrichment steps.
  • PBS containing Ca2+ or Mg2+ is not recommended, except when required in DNAse treatment (as specified in protocol).
  • Do not use buffers or additives (i.e. FCS) containing biotin since this may reduce efficiency of depletion.
  • Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads do not settle at the bottom of the tube.
  • Follow the magnet recommendations to ensure a successful isolation.

Protocol

Dynabeads Washing Procedure

Dynabeads should be washed before use.

  1. Resuspend the Dynabeads in the vial.

  2. Transfer the desired volume of Dynabeads to a tube.

  3. Add the same volume of Buffer 1, or at least 1 ml, and mix.

  4. Place the tube in a magnet for 3 min and discard the supernatant.

  5. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1 as the initial volume of Dynabeads (step 2).

Sample Preparation

As the enriched DC population is intended for further isolation of subpopulations by flow sorting we advise running single cell suspensions of spleen or lymph node cells through a density gradient to remove platelets, cell debris and high density cells (2).

Recommended Protocol for Preparing Starting Sample for Enrichment.

  • This protocol is based on isolation from a maximum of 5 spleens.

  1. Remove the spleens or lymph nodes from recently killed mice and transfer to a 50 ml tube containing 30 ml room tempered Buffer 2 supplemented with 120 IU/ml DNAse.

  2. Crush the tissues through a 70 μm cell strainer into a new tube. Flush the cell strainer with the initial buffer to wash out all cells.

  3. Incubate the tube for 15 min at room temperature (RT) with tilting and rotation.

  4. Add 0.2 ml of 0.5 M EDTA to each tube to dissolve any DC-T cell aggregates.

  5. Mix and incubate for 5 min on a roller at RT with tilting and rotation. Perform further cell processing on ice with cold buffers.

  6. Filter the cells through a new 70 μm cell strainer into a new 50 ml tube and flush the strainer with Buffer 1.

  7. Fill the tube with Buffer 1.

  8. Centrifuge at 300 x g for 10 min at 2-8°C.

  9. Discard the supernatant and resuspend the cell pellet in 5 ml of Buffer 1.

  10. Transfer the cell suspension and layer carefully on top of 4 ml density gradient medium in a 15 ml tube.

  11. Centrifuge at 1700 x g for 10 min at 2-8°C with slow acceleration and no brakes.

  12. Carefully transfer the leucocyte layer to a new tube.

  13. Fill the tube with Buffer 1.

  14. Centrifuge at 300 x g for 10 min at 2-8°C.

  15. Count the cells and resuspend in Buffer 1 to a concentration of 1 x 108 cells/ml.

Enrichment of Mouse DCs

This protocol is based on enrichment from 1 x 10 7 leucocytes. It is scalable from 1 x 10 7-5 x 10 8 cells, (see table 1).

Keep the cells and buffers cold (2-8°C) during the whole process.

  1. Transfer 100 μl (1 x 107) leucocytes in Buffer 1 to a tube.

  2. Add 20 μl of Antibody Mix.

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

  4. Wash the cells by adding 2 ml Buffer 1. Mix well by tilting the tube several times and centrifuge at 300 x g for 10 min at 2-8°C. Discard the supernatant.

  5. Resuspend the cells in 900 μl Buffer 1.

  6. Add 100 μl pre-washed Depletion MyOne SA Dynabeads.

  7. Incubate for 15 min at 2-8 °C with gentle tilting and rotation.

  8. Resuspend the bead-bound cells by thorough pipetting or vortex for 5 secs.

  9. Add 1 ml Buffer 1.

  10. Place the tube in the magnet for 3 min.

  11. Transfer the supernatant to a new tube. The supernatant contains the DC enriched cell population.

Downstream Applications

The DC enriched cell population can be used for further isolation of DC subpopulations to high purity using flow sorting.

Table 1. Volume requirements for mouse DC enrichment.

  Working volume per 1 x 107 leucocytes Working volumes per
1 x 107 leucocytes 1 x 108 leucocytes
Cell volume (step 1)100 μl1 ml
Antibody Mix (step 2)20 μl200 μl
Washing (step 4)2 ml20 ml
Resuspension (step 5)900 μl9 ml
Depletion MyOne SA Dynabeads
(step 6)
100 μl
1 ml
Volume added before magnet (step 9)1 ml10 ml
Dynal MPC recommended MPC-L/MPC-15MPC-50

When working with other cell numbers, scale up all reagents and volumes accordingly. As a rule of thumb, up to 5 x 10 7 leucocytes can be processed in a single 15 ml tube and up to 2 x 10 8 leucocytes can be processed in a single 50 ml tube.


General Information

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

Description of Materials

Dynabeads FlowComp™ are uniform, superparamagnetic beads (2.8 μm in diameter). Supplied at a concentration of approx. 1 × 109 beads (10 mg) per ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3) as preservatives.

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. Shortman K and Liu Y (2002) Mouse and human dendritic cell subtypes. Nature Rev. Imm. 2:151-161.

  2. Henri S et al (2001) The dendritic cell populations of mouse lymph nodes. J. Imm. 167: 741-48.

  3. Vremec D et al (2000) CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Imm. 164: 2978-86.

  4. Baban B et al (2005) A minor population of splenic dendritic cells expressing CD19 mediates IDO-dependent T cell suppression via type I IFN signaling following B7 ligation. Int. Imm. 17(7): 909-19.
114.29D.indd   Rev 002     5-May-2007

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