Isolation of CD34+ cell populations is important for a wide variety of research areas, including studies of hematological malignancies and the homing, survival, migration and differentiation of hematopoietic progenitor cells and endothelial progenitor cells, etc. The CD34 antigen is a single chain transmembrane glycoprotein expressed on human hematopoietic progenitor cells, endothelial progenitor cells, vascular endothelial cells, embryonic fibroblasts, and some cells in fetal and adult nervous tissue. CD34+ cells can be rapidly and efficiently enriched using the CD34 MicroBead Kit which is also known as the Direct CD34 Progenitor Cell Isolation Kit. The CD34 MicroBead Kit contains FcR Blocking Reagent and MicroBeads that are conjugated to monoclonal mouse anti-human CD34 antibody for magnetic labeling of CD34+ cells.
The basic principal behind the CD34 MicroBead Kit is quite straightforward and involves a single-step labeling. First, cells are magnetically labeled with CD34 MicroBeads. Then, the cell suspension is loaded onto a MACS column (available in a variety of sizes based on cell density: MS, LS, or XS columns) which is placed in the magnetic field of a MACS separator (large, free-standing magnets specific to column size). The magnetically labeled CD34+ cells are retained within the column. The unlabeled cells are eluted as the “negative” fraction; this cell fraction is thus depleted of CD34+ cells. After removing the column from the magnetic field, the magnetically retained CD34+ cells can be eluted as the positively selected cell fraction. The protocol also includes several wash steps. An additional advantage to using the direct MicroBeads (as compared to an indirect labeling system) is that there are fewer washing steps involved during the labeling procedure, resulting in reduced cell loss.
This kit is recommended for the isolation or depletion of CD34+ cells from peripheral blood, bone marrow, leukapheresis products or cord blood. Personally, I have used the CD34 MicroBead Kit for enrichment of CD34+ cells from bone marrow and peripheral blood samples. I always performed erythrocyte lysis using RBC lysis buffer (from StemCell Technologies, Inc.) before doing MACS separation. I used a quadro MACS separator, which provides both a magnetic field and a support for the column. The separator is available in different sizes to accommodate various isolation ranges and numbers of columns. For instances, 4 columns can be loaded simultaneously onto a quadro MACS Separator. I generally use an LS column which can be loaded with 2 x 109total cells and is more suitable for my initial sample volumes. I prefer to filter cell suspensions through the pre-separation filter (30 µm nylon mesh filters, Miltenyi Biotec, Cat. no 130-041-407) before loading the samples onto the column in order to remove cell clumps. This can improve the separation, especially when isolating very rare cells. If samples are not fresh, I use the Dead Cell Removal Kit (Miltenyi Biotec Cat. no 130-090-101) for eliminating dead cells prior to MACS separation. To check the purity, a fraction of the isolated CD34+ cells can be labeled with a fluorescent anti-CD34 antibody for fluorescence activated cell sorting. So far, I have had very good results using this kit combined with my experimental procedures and I am consistently getting >95% purity. Usually, I achieve 2 log reductions in the number of CD34+ cells as compared to the number of mononuclear cells loaded onto the column. I used these CD34+ cells for in vitro culturing to analyze the effect of various tyrosine kinase inhibitors on downstream targets and subsequently for Western blot analysis, qPCR analysis and cell cycle analysis. The CD34+ cells isolated from this kit can also be used for studies on hematopoiesis, differentiation, colony formation, and surface marker expression.
Although expensive, this kit provides an efficient and reliable method of cell separation. The initial purchase cost of the separator is high though you need to buy it only once. Columns cannot be reused and on-going replacement of the columns and the MicroBeads is reasonably expensive. Although the system is quite expensive, the only method that might displace such efficiency is Flow Cell Sorting, which is unaffordable for the majority of laboratories. Overall, the kit is very easy to use and very effective and I highly recommend it for biologically relevant experiments.