实验概要
Enrich untouched Dendritic Cells (DCs) by depleting T cells, B cells, monocytes/ macrophages, NK cells, erythrocytes and most granulocytes from blood mononuclear 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. This kit provides high recovery of lineage specific markers (Lin-) CD4 cells and is therefore suitable for further isolation of any DC subpopulation, such as myeloid and plasmacytoid DCs.
实验原理
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
主要试剂
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 ).
Lymphoprep®
实验步骤
1. 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 transferred from the vial in step 2.
2. Sample Preparation
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.
3. 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. 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.
4. Downstream Applications
The DC enriched cell population can be used for further isolation of DC subpopulations to high purity using flow sorting.
注意事项
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.
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