Which columns and magnets or separation program can be used with the Blood Dendritic Cell Isolation Kit II, human?
The Blood Dendritic Cell Isolation Kit II, human is a magnetic labeling system for the concurrent isolation of plasmacytoid and myeloid DCs from human PBMCs. The isolation is performed in a two-step separation with MACS® Technology. First, B cells and monocytes are magnetically labeled and depleted using a cocktail of CD19 and CD14 MicroBeads. Subsequently, the pre-enriched DCs in the non-magnetic flow-through are magnetically labeled and further enriched using a cocktail of antibodies against the DC markers CD304 (BDCA-4/Neuropilin-1), CD141 (BDCA-3), and CD1c (BDCA-1).
It is recommended to use an LD Column for the depletion step and two MS Columns for positive selection. Using the autoMACS™ Separator or the autoMACS™ Pro Separator, it is recommended to use deplete05 and posseld2 for depletion and positive selection, respectively.
How can the purity of DCs isolated with the Blood Dendritic Cell Isolation Kit II, human be evaluated?
The Blood Dendritic Cell Enumeration Kit II, human is the most convenient way for analysis. It includes pre-mixed, ready-to-use cocktails for DC detection and for isotype control and allows exclusion of dead cells for more reliable results.
The Blood Dendritic Cell Enumeration Kit II, human was developed on the basis of unique BDCA markers. It contains the markers CD1c (BDCA-1), CD141 (BDCA-3), and CD303 (BDCA-2) for the characterization of different blood DC subsets. To allow the exclusion of B cells and monocytes from DC analysis, the antibody cocktails contain antibodies against CD19 and CD14, respectively.
Is it recommended to generate DCs from CD14+ cells or from CD34+ cells?
Generally, both strategies can be used to generate DCs . However, the choice for one of the strategies should be made, depending on the planned experiment or application, since the obtained DCs differ in certain aspects. Monocyte-derived DCs can be loaded very efficiently with antigen. In contrast, CD34+ progenitor cell derived DCs are more suitable for retroviral transduction due to their proliferative activity.
Can CD14+ or CD34+ cells directly be taken into culture after isolation with MACS® Technology?
MACS® MicroBeads do not affect the viability of the isolated cells. For subsequent cell culture, the cells on the column can be washed with culture medium instead of buffer. The medium can then also be used for the elution of the cells from the column, eliminating any further washing steps.
What is the frequency of dendritic cells (DC) in human peripheral blood and which cell numbers can be expected when using the Blood Dendritic Cell Isolation Kit II, human?
DCs typically represent 0.1–1% of the circulating PBMCs. When starting with 50 mL of blood, an average number of 15,000 to 150,000 purified DCs can be obtained using the Blood Dendritic Cell Isolation Kit II, human.