Isolation of CD34+ Cells from Mononuclear Cells (MNCs) from Human Bone Marrow, Peripheral Blood or Cord Blood
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实验试剂
Mixer allowing both tilting and rotation
Buffer: PBS (without Ca2 and Mg2 ) w/0.1% BSA and anticoagulant, pH 7.4
Culture Medium: e.g. RPMI 1640 with 2% Fetal Calf Serum (FCS)
Density gradient medium for MNC (1.077g/l).
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, or at least 1 ml, and mix.
4) Place the tube in a magnet for 1 min and discard the supernatant.
1) Resuspend the prepared cell sample thoroughly with a narrow pipette.
2) Add 100 μl Dynabeads® to the prepared sample and vortex 2-3 seconds.
3) Incubate at 2 - 8°C for 30 min. with gentle tilting and rotation.
5) Place the tube in a magnet for 2 min and discard the supernatant.
8) Resuspend the bead-bound cells in 100 μl Buffer.
9) Add 100 μl DETACHaBEAD. (Never use less than 100 μl DETACHaBEAD.)
10) Add 2 ml Buffer and vortex 2-3 seconds to enhance detachment of beads from the cells.
11) Place the tube in a magnet for 2 min.
14) Resuspend the cells in Buffer or Culture Medium.
注意事项
