Human DC Enrichment Kit
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实验原理
实验试剂
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 ).
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.
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.
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.
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.
10) Place the tube in the magnet for 3 min.
11) Transfer the supernatant to a new tube.
注意事项
