Q：What is the principle of immunomagnetic cell sorting?

A：Immunomagnetic cell sorting technology is based on the principle of antigen antibody specific binding in immunology: Magnetic particles are used as carriers to coat them with antibodies or affinity ligands to form immunomagnetic composite particles. When it is incubated with mixed cells, the antibody on the surface of magnetic particles will specifically bind to the antigen determinant on the cell surface. Cells are labeled with magnetic composite particles. Under the effect of the external magnetic field, the cells connected with the antibody and the magnetic beads will stay in the magnetic field. The cells that do not express this antigen cannot bind to the specific antibody on the surface of the magnetic bead, so they do not have magnetism and cannot remain in the magnetic field. Thus, the target cells can be separated with high purity.

Q：What is the relationship and difference between positive sorting and negative sorting?

A：The immunomagnetic cell sorting system can be divided into positive sorting and negative sorting according to the different cell types marked in the sorting process.

      Positive sorting is to directly separate the target cell subpopulation from the cell suspension. Only one antibody (corresponding to the target cell) is required to participate in the sorting process to achieve high purity sorting. However, because the cells are connected with the magnetic beads, it may stimulate the cells.

      Negative sorting is a method to separate and remove non target cells from multi cell suspension to obtain target cells. The sorting process requires the participation of multiple antibodies (corresponding to non target cells). Because the target cells are not connected with the magnetic beads, there is no stimulation to the cells, but the non target cells may not be fully removed, resulting in low sorting efficiency and purity.

Q：Buy positive sorting kit or negative sorting?

A：In order to select suitable products, it is necessary to integrate the characteristics of the test purpose and sorting method. The positive sorting kit has high sorting purity and specificity, but the cells are connected with magnetic beads; the cells of the negative sorting kit are not connected with the magnetic beads, and the sorting purity may be slightly lower than that of the positive sorting kit. If you want to remove certain types of cells through magnetic bead sorting, it is recommended to use a positive sorting kit; if you want to obtain the target cells for later culture, it is recommended to select the negative selection kit.

Q：How many cells can be sorted out with a 10 test sorting kit?

A：1 test in the kit can process1×10⁷PBMCs，10 tests can process 1×10⁸PBMCs. The sorting efficiency of the kit can reach more than 90%. However, the specific number of cells that can be sorted needs to evaluate the theoretical number of target cells in PBMC.

Q：How to select PBMC for cell sorting? Fresh or Frozen?

A：Fresh PBMC can ensure good cell state and sorting effect. It is recommended to use fresh PBMC to sort subtypes of cells. If fresh PBMC is difficult to obtain, frozen PBMC can also be selected, but the separation purity and recovery are difficult to ensure.

Q：Which tissues and organs can the sorting kit be used for?

A：Single cell suspension is the basis of cell sorting with sorting kit. Theoretically, it can be used for cell sorting from any cell suspension rich in target cells. At present, only PBMC and spleen mononuclear cell sorting have been verified. If it is required to be used for the sorting of special organizations, the conditions shall be explored and verified by yourself.

Q：Can the sorting kit of our company be used with the magnetic rack of other manufacturers?

A：The sorting reagent boxes of our company all use nano magnetic beads, which requires high magnetic field strength of the magnetic frame. It is recommended to use our company's special magnetic frame for cell sorting. If use magnetic racks from other manufacturers, it is necessary to explore and verify the separation conditions.

Q：What effect does temperature have on the separation effect?

A：The kit recommends cell sorting at 4 ℃. Cell sorting at 4 ℃ can maintain the original state of cells in a short time. If the sorting temperature is too high, such as 37 ℃, some metabolic reactions of cells may be activated, resulting in poor viability of the sorted cells. It is suggested that cell sorting should be carried out in strict accordance with the test conditions recommended by the kit.

Q：Can the cells obtained through the positive sorting kit be detected by flow cytometry?

A：The cells obtained by positive sorting are connected with magnetic beads. The magnetic beads used are nano magnetic beads. Theoretically, it does not affect the detection of flow cytometry. If you have doubts, you can try to use the method of culture plus repeated blowing and suction to separate the cells from the magnetic beads before flow detection. It should be noted that the selection of flow antibody should strictly refer to the product description.

Q：Which sorting method is used to obtain immune cells of each subtype?

A：Various subtypes of immune cells provided by our company are sorted by immunomagnetic negative method. It is obtained from freshly isolated mononuclear cells. All cells have been tested for purity, viability and other indicators. Although the purity and viability of cells of different subtypes are different, they can all reach more than 90%.

Q：How to cultivate T lymphocytes?

A：T lymphocytes belong to primary cells and cannot be directly cultured in vitro. If you want to maintain and proliferate them in vitro, you need to use anti-CD3 and anti-CD28 antibodies to jointly stimulate cells and culture them in the presence of IL-2 cytokines.