Two images showing the Sep-Mate tube before and after separation.
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Our lab investigates how the diversity of innate immune system genes impacts immune system function. Specifically, we study the clonal diversity arising from differential expression of NK cell receptors both in individuals and in human populations. DNA extraction from PBMCs and flow cytometry with purified NK cells are two experimental tools that we commonly use to answer specific questions in this field. As a result, we often have need to isolate PBMC and NK cells from patient or donor samples. Isolated DNA is typically used for kill-cell immunoglobulin-like (KIR) and HLA class I gene sequencing and purified NK cells are studied using flow cytometry to better understand how specific genotypes translate into receptor phenotypes.
To isolate NK cells, our lab uses a standard density gradient centrifugation method using Ficoll® to isolate PBMCs from donor blood samples, then isolates NK cells from PBMCs using immunomagnetic cell separation. Density gradient centrifugation requires careful layering of diluted blood on the Ficoll, followed by a centrifugation step of about 30 minutes. The PBMCs are harvested by removing the cells from the interface of the Ficoll and the plasma layer using a pipette. The PBMCs are then washed twice in PBS + FBS, and then the NK cells are isolated using magnetic cell separation. This process is laborious (up to 45 min) and takes considerable practice to collect all of the PBMCs without contamination with either Ficoll or the plasma layer. Missteps in separation are common and can often result in loss of valuable and unique clinical samples. We have recently started using the SepMate system that makes isolation of PBMCs not only more straightforward, but also considerably less time-consuming.
Cell separation for subsequent flow cytometry analysis and for DNA isolation and sequencing
I typically process 50-100 ml of blood from human or chimpanzee donors
For each donor sample, 15 ml of Ficoll is added to the Sep-Mate tube such that it fills up the space below the insert in the specially designed 50 ml tube. Each blood sample is then split into 15 ml aliquots and diluted 1:1 with FBS and PBS. This mixture is then added to the Sep-Mate tube. Unlike with traditional Ficoll separations, this can be done quickly as the insert in the Sep-Mate tube prevents the blood from mixing with the Ficoll. Each sample is then spun for 10 minutes (with the brake on) and the PBMC layer is collected simply by pouring the entire top layer into a collection tube. We have found that using this system allows us considerable time-savings (10 minutes as opposed to 30 minutes centrifugation - and you can leave the brake on, saving an additional 2-3 minutes). The time-savings adds up with multiple samples, since the layering and harvesting occurs much more quickly and easily than in the standard protocol.
Be sure to fill the sep-mate tube with 15ml of Ficoll so that it completely fills the bottom well of the tube. Using less than this amount can lead to white blood cells being deposited on the plastic insert.
SepMate-50 uses apparently simple technology to solve a common problem for our lab. We have been able to save time and cut down sample loss due to poor PBMC separation. We are able to recover as many NK cells as with traditional Ficoll separations. I did notice that there were some red blood cells on the surface of the insert, but I did not see any red blood cell contamination in my PBMC pellet after the cells were washed. One small downside is that the maximum volume that can be used in the tube is 17.5 ml of blood (35 ml after dilution) meaning I needed to use 4 or more tubes are required for one donor sample. That being said, our time savings likely make up for this slight inconvenience – for example we are able to start NK cell isolations earlier than before, which is great since our blood samples often arrive late in the afternoon.
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