A method for sorting, isolating, and purifying primary cells by immunomagnetic beads coated with a specific antibody or an anti-IgG antibody:
Very high purity primary cells of interest can be isolated from complex primary cell mixtures in a matter of minutes.
Very pure primary cell populations can be isolated with coating-specific or anti-IgG antibodies with excellent recovery and survival. The purity of primary cells can range from 95% to 99.9% depending on the cell frequency and the level of marker expression.
Fundamental:
The coated specific antibody magnetic beads have been specifically bound to the corresponding molecules on the surface of the primary cells, or the anti-IgG antibody magnetic beads are bound to specific antibodies that have previously specifically bound to the cell surface molecules. The magnetic beads carry the cells bound to them on the separation column/test tube to achieve positive cell separation or negative cell separation.
Basic classification:
The immunomagnetic beads method is divided into a positive cell separation method and a negative cell method:
Positive Cell Isolation - Magnetic beads-bound cells are the cells to be isolated. Negative cell separation method - magnetic beads bind unwanted cells, and cells freed from the supernatant are desired cells.
In general, the negative cell separation method uses a larger amount of magnetic beads than the positive cell separation method.
The basic steps:
1. Collect the cells to be separated by centrifugation, and fully suspend the cells (0.5 ml/1×108 cells) with a small amount of PBE incubation solution (0.5% BSA, 0.08% EDTA pH 7.2 PBS, vacuum filtration and liquid gas). The specific antibody magnetic beads or anti-IgG antibodies were coated and incubated at 4 ° C for 30 minutes.
2. Wash the cells once with 20 times volume of PBE, then add PBE (0.3ml/1×108 cells) to fully suspend the cells, then add the corresponding micro-adhesive coated ultra-magnetic beads, mix and set 8~15°C. Incubate for 10 to 15 minutes.
3. Install the separation column into the magnetic field, add 0.5ml of PBE, and naturally drain it under the action of gravity to pretreat the separation column.
Suggest:
1. The whole process of separating cells is completed in a clean bench.
2. Ultra-microbeads and tiny magnetic field systems are suitable for 106-107 cell separation.
3. The separation column can only be applied once.
4. Try to remove dead cells.
5. When the cell suspension is added to the separation column, the pipette should be extended to the bottom wall and added to avoid the inflow of the cell suspension along the tube wall.
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