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
- 20 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.
- 4 ml Antibody Mix for DC kit
The Antibody Mix contains an optimized mixture of biotinylated monoclonal antibodies against CD3, CD14, CD16, CD19, CD56 and Glycophorin A supplied in phosphate buffered saline (PBS), pH 7.4, containing 0.02% sodium azide. The kit will process 2 x 109 mononuclear cells.
Additional Materials Required
- Magnet: (Dynal® MPC™), MPC-L for 1–5 ml samples, MPC-15 for 1–15 ml samples and MPC-50 for 15–50 ml samples.
- 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 pH 7.4 (without Ca2+ and Mg2+).
- It is critical to keep the buffers cold (2–8˚C) during the enrichment steps.
- PBS containing Ca2+ or Mg2+ is not recommended.
- 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.
Dynabeads® should be washed before use.
- Resuspend the Dynabeads® in the vial.
- Transfer the desired volume of Dynabeads® to a tube.
- Add the same volume of Buffer 1, or at least 1 ml, and mix.
- Place the tube in a magnet for 3 min and discard the supernatant.
- Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume transferred from the vial in step 2.
Preparation of MNC from Buffy Coat to Obtain Low Platelet Numbers
1. Dilute 10 – 18 ml buffy coat with Buffer 2 to a total volume of 35 ml at room temperature.
2. Add the diluted buffy coat on top of 15 ml of Lymphoprep.
3. Centrifuge at 160 x g for 20 minutes at 20°C. Allow to decellerate without brakes.
4. Remove 20 ml of supernatant to eliminate platelets.
5. Centrifuge at 350 x g for 20 minutes at 20°C. Allow to decellerate without brakes.
6. Recover MNC from the plasma/Lymphoprep interface and transfer the cells to a 50 ml tube.
7. Wash MNC once with Buffer 1 by centrifugation at 400 x g for 8 minutes at 2–8°C.
8. Wash MNC twice with Buffer 1 by centrifugation at 225 x g for 8 minutes at 2–8°C and resuspend the MNC at 1 x 108 MNC per ml in Buffer 1.
Enrichment of DCs
- This protocol is based on enrichment from 1 x 107 leucocytes (MNC). It is scalable from 1 x 107–2 x 109 cells, (see table 1).
Keep the cells and buffers cold (2–8°C) during the whole process.
- Transfer 100 μl (1 x 107) leucocytes in Buffer 1 to a tube.
- Add 20 μl of Antibody Mix.
- Mix well and incubate for 20 min at 2–8°C.
- 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.
- Resuspend the cells in 900 μl Buffer 1.
- Add 100 μl pre-washed Depletion MyOne SA Dynabeads®.
- Incubate for 15 min at 2–8°C with gentle tilting and rotation.
- Resuspend the bead-bound cells by thorough pipetting or vortex for 5 secs.
- Add 1 ml Buffer 1.
- Place the tube in the magnet for 3 min.
- Transfer the supernatant to a new tube.
The supernatant contains the DC enriched cell population.
Table 1. Volume requirements for DC enrichment.
|Working volumes Working volumes per 1 x 107 leucocytes||Working volumes per 5 x 108 leucocytes|
|Cell volume (step 1)||100 μl||5 ml|
|Antibody Mix (step 2)||20 μl||1 ml|
|Washing (step 4)||2 ml||35 ml|
|Resuspension (step 5)||900 μl||10 ml|
|Depletion MyOne SA Dynabeads® (step 6)||100 μl||5 ml|
|Volume added before magnet (step 9||1 ml||15 ml|
|Dynal® MPC recommended||MPC-L/MPC-15||MPC-50|
When working with other cell numbers, scale up all reagents and volumes accordingly.
The DC enriched cell population can be used for further isolation of DC subpopulations to high purity using flow sorting.
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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