We isolate NK cells using a kit from Stem Cell Technologies but since the proportion in PBMC is relatively low we're looking for a method to enrich them before using the kit. Has anyone used this or another method that might be helpful?
Article Isolation of human NK cells by density gradient centriguation
Dear Anne, in our lab we routinely isolate NK cells from whole blood, to do so we first enrich in PBMCs (and therefore in NK cells) by performing a density gradient centrifugation using Ficoll-PaqueTM Plus (GE Life Sciences) centrifugation. In this case, it is important to separate in other tube 0.5 ml per 20 ml of whole blood to use it later with the Stem Cell kit. Then, we use Stem Cell kit by mixing PBMCs obtained from Ficoll-Paque density gradient centrifugation with the previously separated volume of whole blood . Finally, we perform an additional step of centrifugation with Ficoll-Paque. I hope this information is useful for you!
Interesting. So you use the RosetteSep kit so the rbc help to remove the non NK cells?
Exactly. The reagents in the kit bind to red blood cells and other non NK cells and deplete the cell suspension from them, leaving NK cells untouched.
Hello
For suspension PBMCs by density gradient centrifugation: buffy coats were diluted by adding an equal volume of PBS; then 2 volumes of the diluted buffy coat were carefully added on top of one volume of LymphoprepTM (Axis-Shield), without mixing; tubes were centrifuged at 800g for 30min at 23ºC and with no brake. PBMCs were collected from the interface and were washed with cold PBS by centrifuging at 300g for 10min at 4ºC for as many times as necessary to remove platelets. and , NK cells were purified by negative selection using the EasySep® human NK cell enrichment kit (StemCell Technologies), according to manufacturer’s instructions. Briefly, PBMCs were resuspended at 5×106cells/mL in 0.5% BSA/2mM EDTA/PBS in a 5mL polystyrene tube. Then, the EasySep® Human NK cell Enrichment cocktail was added at a concentration of 50µL/mL of cells. Cells were mixed and incubated at room temperature for 10min, magnetic particles were added at 100µL/mL cells and incubated at room temperature for 5min. Then, 0.5%BSA/2mM EDTA/PBS was added to a total volume of 2.5mL, and the suspension was gently mixed. The tube was placed into a purple EasySep® magnet and set aside for 2.5min. The magnet and tube were then inverted in one continuous motion, pouring off the desired fraction into a new tube. In order to recover more cells, the magnet was returned to an upright position, 2.5mL of 0.5%BSA/2mM EDTA/PBS were added, cells were mixed and set aside for another 2.5min and the new fraction was collected to the same tube.
Good Luck
Dear Raul German Spallanzani
I would like to isolate NK cells using your mentioned technique and the RosetteSep Isolation Kit from 25 ml whole blood. So could you guide me on the details of the protocol of how you isolated the PBMCs (speed, ratio) and if any dilution was done? Also, if I would like to check the purity for NK cells what is the best marker that I should go for ?
Thanks so much for your help in advance.
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