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        Human DC Enrichment Kit

        互联网

        1161

        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

        实验试剂

         

        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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