Isolating Plasmacytoid Dendritic cells (pDCs) from peripheral blood has always been very difficult; pDCs make up a very low percentage of total circulating peripheral blood mononuclear cells (PBMCs). In addition, their possible activation from antibodies binding to receptors on their surface adds another challenge; this feature also makes negative selection the most feasible way of isolating pDCs for subsequent culture and functional assays. Stemcell Technologies’ EasySep® Human Plasmacytoid DC Enrichment kit is the first kit of its kind that allows for an end user to isolate very pure populations of pDCs (consistently in excess of 97 % pure CD303+ HLA-DRhigh
pDCs) from PBMCs.
The protocol is very simple. Once PBMCs have been isolated by density gradient centrifugation, a series of reagents are added and incubated in a specific order. The protocol is very easy to follow and unlike column based magnetic cell separation techniques, there are no lengthy or repetitive wash steps to be completed in order to remove non-specific cell types. Once the cells have been incubated with the blocking reagent, an antibody cocktail (to bind all unwanted cell types using a clever mixture of specific antibodies) and magnetic beads, the sample tube is simply then applied to the EasySep® magnet for 5 minutes. After this, all the unwanted labeled cells are drawn to the outside of the tube leaving the untouched pDCs in suspension, which can then be simply poured into a fresh collection tube. The supplied protocol is not only fundamentally very simple to follow but also extremely quick and allows the user to go from samples of whole blood to enriched populations of pDCs in just a few hours.
My research is centred around examining the different changes that occur in both myeloid and lymphoid DCs resident in peripheral blood when they are stimulated to undergo maturation. Unlike other kits, this negative selection protocol allows me to isolate very pure populations of untouched, unstimulated, naive pDCs from whole blood in a very short amount time, which has the knock on affect of increasing their viability for subsequent culture and improves the quality of results we see from our qRT-PCR and ChIP assays.
Figure 1. Data above show a sample of my own experiments. Prior to enrichment, the percentage of pDCs in total PBMC is approximately 0.1 % (upper row), however following enrichment, a population of pDCs in excess of 97% pure can be obtained (lower row). pDCs identified using surface marker staining for CD303 and HLA-DR.
In summary, as long as the easy to follow and well-written protocol is followed accurately, this kit consistently yields very high populations of ‘untouched’ pDCs which can then be cultured and used for many different downstream experiments. The kits themselves, whilst expensive are extremely effective and can also be stored for long periods of time at 2- 8°C, much longer than other bead-based isolation kits. The initial high cost of the EasySep® magnet aside, this is possibly the most effective and efficient kit for isolating human pDCs presently on the market.
EasySep® Human Plasmacytoid DC Enrichment Kit from StemCell Technologies
The kits are extremely effective and can also be stored for long periods of time at 2- 8°C, much longer than other bead-based isolation kits.
The Bottom Line
Possibly the most effective and efficient kit for isolating human pDCs presently on the market.