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MACS Handbook
Hematopoietic stem cells (HSC) are tissue-specific adult stem cells capable of differentiating into all blood cell types to ensure homeostasis of blood throughout life.
Research on murine HSCs dates back to late the 1940s early 1950s, when it was discovered that sub-lethally irradiated mice could be rescued from hematopoietic failure by injecting cells from blood-forming organs, such as the bone marrow. This finding lead to the idea that cells in bone marrow with repopulation capacity could be further characterized and quantified in transplantation models. In early experiments, decreasing numbers of bone marrow cells were injected into recipient mice to determine the lowest cell number need to fully reconstitute hematopoiesis. Further experiments transplanting sub-radioprotective doses of bone marrow cells into mice showed that these mice developed colonies of hematopoietic cells in their spleens. The number of colonies found revealed a clear correlation to the amount of bone marrow cells originally injected. The advent of fluorescence-activated cell sorting technology and monoclonal antibodies then finally lead to the identification of surface markers which are used to identify mouse hematopoietic stem cells.
Related PDFs:
Stem Cell Quick Guide (brochure)
At a glance: HSCs in bone marrow
| HSC source | Frequency | Marker Expression | Function |
|---|---|---|---|
| Bone marrow | 0.01% | Lin– Sca-1+ CD117+ CD48– CD150+ | Hematopoiesis |
Mouse HSCs are characterized and/or isolated using several surface markers. Firstly, mouse HSCs lack the expression of lineage-specific markers (ie., are lin–), and are positive for c-kit (CD117) and Sca-1. More recently, the HSC population was further narrowed down by including CD150 and CD48, resulting in the marker profile Lin–c-Kit+Sca-1+CD48–CD150+.
Miltenyi Biotec has created dedicated application protocols to work with and analyze HSCs.
Murine HSCs are usually isolated from bone marrow prepared from the tibia of the hind leg.
Miltenyi Biotec has developed products to pre-enrich lineage-negative cells containing HSCs from mouse bone marrow cells using the MACS® Cell Separation Technology.
For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic Cell Separation .
MACS Handbook:
Magnetic cell separation
At a glance: Kits and reagents for the separation of HSCs from bone marrow
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Bone marrow | Positive selection of target cells | Anti-Sca-1 MicroBead Kit (FITC), mouse | ||
| Bone marrow | Positive selection of target cells | CD105 MultiSort Kit (PE), mouse | ||
| Bone marrow | Positive selection of target cells | No | CD117 MicroBeads, mouse | |
| Bone marrow | Depletion of non-target cells | Direct pre-enrichment of lineage negative cells | Yes | Direct Lineage Cell Depletion Kit, mouse |
| Bone marrow | Depletion of non-target cells | Stringent two-step pre-enrichment of lineage negative cells | Yes | Lineage Cell Depletion Kit, mouse |
Quick, one-step separation of lineage-negative cells. Untouched lineage-negative cells were isolated from a single-cell suspension of mouse bone marrow using the Direct Lineage Cell Depletion Kit, mouse and a MidiMACS™ Separator with an LS Column. Cells were fluorescently stained with Lineage Cell Detection Cocktail-Biotin and Anti-Biotin-APC and analyzed by flow cytometry using the MACSQuant® Analyzer. To evaluate the LSK (Lin–Sca-1+c-kit+) fraction, cells were further stained with CD117-PE (c-kit) and Anti-Sca-1-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
HSCs can be further separated from lineage negative (Lin–) cells using specific MACS MicroBeads, like the CD117 MicroBeads, mouse or the Anti-Sca-1 MicroBead Kit.
CD117 MicroBeads isolate murine progenitor cells. CD117 (also known as c-kit, steel factor receptor, or stem cell factor receptor) encodes a 145 kD cell surface glycoprotein belonging to the class III receptor tyrosine kinase family. It is expressed on the majority of hematopoietic progenitor cells, including multipotent hematopoietic stem cells and committed myeloid, erythroid, and lymphoid precursor cells. In addition to their hematopoietic cell differentiation potential, CD117+ stem cells from murine bone marrow are reportedly capable of differentiating into smooth muscle cells, myocytes, and endothelial cells in vivo. CD117 is also expressed on a few mature hematopoietic cells, like mast cells.
Related PDFs:
MACS Cell Separation - Select the best (brochure)
Stem Cell Quick Guide (brochure)
Related videos:
At a glance: Dedicated antibody cocktail for flow cytometry analysis of HSCs
| Markers in cocktail | Isolation kit or reagent used | Comment | Product |
|---|---|---|---|
Biotin-conjugated monoclonal antibodies against CD5, CD11b, CD45R (B220), Anti-7-4, Anti-Gr-1 (Ly-6G/C), and Anti-Ter-119. | Lineage Cell Depletion Kit, mouse | Requires the use of a secondary antibody such as Anti-Biotin-FITC, Anti-Biotin-PE, or Anti-Biotin-APC | Lineage Cell Detection Cocktail-Biotin, mouse |
Hematopoietic stem cells are routinely analyzed by flow cytometry based on various extracellular and intracellular markers. The Lineage Cell Detection Cocktail-Biotin, mouse simplifies the flow cytometry analysis of cells before and after cell separation using MACS Technology. All antibodies are optimally titrated to be simply added to an aliquot of a cell fraction before analysis.
Miltenyi Biotec also offers a wide range of unique and standard monoclonal antibodies for research of HSCs and progenitor cells, including CD117, Anti-SCA-1, CD150, and CD48. Fluorochrome-conjugated MACS Antibodies are perfectly suited for the identification, enumeration, and characterization of HSCs, which can be combined into panels tailored to specific research needs. An online tool to quickly construct the right multicolor flow cytometry panel for each research project can be access via the Related Resources panel to the right.
For details about Miltenyi Biotec antibodies and dyes for cell analysis, see chapter Cell Analysis – Reagents.At a glance: Cytokines for the expansion of mouse HSCs
| Use | Comment | Product |
|---|---|---|
| Expansion | Mouse SCF, premium grade | |
| Expansion | Mouse FLT-3-Ligand, research grade | |
| Expansion | Mouse IL-3 IS, premium grade | |
| Expansion | Mouse IL-6, premium grade |
MACS Handbook:
Cell culture
At a glance: Kits and reagents for viral transduction of HSCs
| Use | Comment | Product |
|---|---|---|
| Viral transduction | Does not have to be pre-coated on cell culture surfaces | Vectofusin-1 |
Viral transduction is a fast and efficient method to study gene function, modulate gene expression, or as potential vector for gene correction in gene therapies. The transduction of HSCs is especially interesting because of their potential to permanently cure hematopoietic defects. The modification of HSCs with retroviral vectors often requires the presence of a transduction-enhancing reagent. Polycationic reagents induce aggregation of vector particles and facilitate binding to target cells via electrostatic interactions. Bridging molecules, such as recombinant fibronectin, interact with both vector particle and cell membrane. Transduction performance can be enhanced with centrifugation.
Miltenyi Biotec offers the novel transduction enhancer Vectofusin-1®, a fully synthetic non-toxic cationic amphipathic peptide that supports high transduction levels with low amounts of retroviral vector. When added to culture medium, Vectofusin-1 promotes the entry of several retroviral pseudo-types into target cells.
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