Magnetic Depletion of SSEA-4+ Undifferentiated Embryonic Stem Cells

实验概要

Human  embryonic stem cells can be differentiated into neural-, mesenchymal  and hematopoetic stem cells. This product is intended for the magnetic  depletion of SSEA-4 undifferentiated embryonic stem cells  from a differentiated cell population to increase purity. Isolated  SSEA-4 negative cells are bead- and antibody free and are suitable for  any downstream application. The products can also be used to isolate the  undifferentiated SSEA-4 positive cells, however, SSEA-4 cells will bind to Depletion MyOne SA Dynabeads® and removal of beads after cel  lysis is recommend prior to analysis.

实验原理

SSEA-4 antibodies are biotinylated and bind to the non-differentiated SSEA-4  cells in the mixed cell population. Depletion MyOne SA Dynabeads® are  added and will bind to the antibody labeled cells during a short  incubation. The bead-bound cells are subsequently separated on a magnet.  The remaining untouched and bead-free cells in the supernatant can be  used for any application. If undifferentiated SSEA-4 cell  will be used, gene and protein analysis can be performed after lysis of  the cells. Place the lysed cells in the magnet to remove beads prior to  analysis.

主要试剂

Isolation Buffer: Ca2 and Mg2 free phosphate buffered saline (PBS) with 0.1 % BSA and 2 mM EDTA

Mixer allowing both mixing and tilting.

Magnet (DynaMag™)

实验步骤

1.        Dynabeads® Washing procedure

1)        Resuspend the Dynabeads® in the vial.

2)        Transfer the desired volume of Dynabeads® to a tube .

3)        Add the same volume of Isolation Buffer, or at least 1 ml, and mix.

4)        Place the tube in a magnet for 1 min and discard the supernatant.

5)         Remove the tube from the magnet and resuspend the washed Dynabeads® in  the same volume of Isolation Buffer as the initial volume of Dynabeads®  (step 2).

2.        Preparations

1)        Prepare the Isolation Buffer.

2)        Prepare a single cells suspension of the cells to a concentration of 1 × 10⁷ cells/ml Isolation Buffer.

3.        Isolation Procedure

This protocol is based on 5 × 10⁶ cells and is scalable from 5 × 10⁶ to 5 × 10⁸ cells.

1)        Transfer 500 μl (5 × 10⁶) cells in Isolation Buffer to a tube.

2)        Add 25 μl SSEA-4 Antibody and mix well.

3)        Incubate 15 min at 18-25°C.

4)        Add 1 ml Isolation Buffer and spin cells for 8 min at 300 × g.

5)         Remove the supernatant and resuspend the cell-pellet in 500 μl  Isolation buffer, add 50 μl of pre-washed Depletion MyOne SA Dynabeads®.

6)        Mix well and incubate for 15 min at 18-25°C with gentle tilting and rotation.

7)         Add 1 ml of Isolation Buffer (if the volume from step 5 is greater than  1 ml, add the 1 ml Isolation Buffer after step 8).

8)         Resuspend the bead-bound cells by pipetting > 10 times with a  pipette with a narrow tip opening, (e.g. a 1000 μl pipette tip or a 5 ml  serological pipette).

9)        Place the tube in the magnet for 2 min.

10)    Transfer the supernatant, containing the bead-free SSEA-4 negative cells, to a new tube.

11)    Repeat step 9-10 to remove residual beads.

注意事项

This  product is for research use only. Not for animal or human therapeutic  or diagnostic use. Follow appropriate laboratory guidelines. This  product contains 0.02% sodium azide as a preservative, which is  cytotoxic.

Avoid pipetting by mouth!