實驗概要
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.
實驗原理
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.
主要試劑
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?)
實驗步驟
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.
注意事項
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!
