造血幹細胞の分離

Questions & answers

Is a flow cytometric purity evaluation of CD34+ or CD133+ cells, isolated with MACS® Technology, possible without removing the MicroBeads?

Yes, it is. Due to their extremely small size MACS® MicroBeads neither interfere with flow cytometric analysis nor with microscopic analysis. They are compatible with flow sorting. The antibody used for detection should recognize a CD34 or CD133 epitope that is different from the epitope recognized by the antibody conjugated to the MicroBeads. The antibody clone QBEND/10 used for the CD34 MicroBead Kit, human, and CD34 MultiSort Kit, human, binds to epitope II of the CD34 antigen. For the staining of CD34+ cells isolated with MACS® technology, one should use an antibody against CD34 epitope III, e.g., MACS® control antibodies CD34-FITC, human, CD34-PE, human, or CD34-APC, human (clone AC136).
CD133+ cells separated with the CD133 MicroBead Kit, human, are best stained with CD133/2 (293C3)-PE, human, or with CD133/2 (293C3)-APC, human.

Do CD34 or CD133 MicroBeads interfere with proliferation and differentiation of hematopoietic cells in culture?

No, they do not. Cells can be cultured straight after separation with MACS® Technology. Proliferation and differentiation of hematopoietic cells in different culture systems remain unaffected. However, prolonged storage of cells in EDTA-containing buffer may have a slightly negative effect on cell proliferation. EDTA can be replaced by other supplements, such as 0.6% ACD-A or citrate phosphate dextrose (CPD).

Can MACS® Technology be used for the isolation of CD34 subpopulations?

Yes, it can. When using CD34 MultiSort Kit, human, it is also possible to sort according to other antigens, in addition to the CD34 selection. After isolation of CD34+ cells using CD34 MultiSort MicroBeads, the MicroBeads are enzymatically released from the cells. Thereafter, cells can be labeled and separated using MicroBeads that bind to additional antigens such as HLA-DR, CD38, CD71, CD95, or CD117.

What starting materials can be used for isolation of hematopoietic stem and progenitor cells with MACS® Technology?

Using MACS® Technology, hematopoietic stem and progenitor cells can be isolated from fresh and frozen mononuclear cells of peripheral blood, cord blood, and bone marrow. Cells from leukaphereses harvests can be labeled without prior processing. Detailed protocols for preparation of hematopoietic stem and progenitor cells from different sources are provided with the data sheets.

Contact

Miltenyi Biotec K.K.
TEL +81 3 5646 8910
FAX+81 3 5646 8911
macs@miltenyibiotec.jp
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