When I use a DX5+ Miltenyi kit, the purity is between 50-60%. If I use cell sorting (without any previous separation step), the day after the sorting, NK cells are not functional for cytotoxicity assays (even if they are cultivated with IL-15 1 ng/mL). If I do first DX5+ Miltenyi kit followed by cell sorting, I lose so many cells that I can't do anything.
HI,
we are currently using the mouse NK cell isolation kit II from Miltemyi and it works great.
More than 90% purity and cells are viable for up to 6 days.
We tried the NKp46 kit but it activates the cells.
Good luck with your isolation.
NK cell isolation kit from Miltenyi works fine. For increasing the purity, I prefer going through two rounds of column elution. It increases the purity by great extent.
Cheers
Hi! Depending on your mouse strain the DX5 kit might not be the best choice considering how it might pick up B cell contamination. If you can, I would switch to the NKp46+ isolation kit from Miltenyi which works like a charm (NKp46 is a much more specific NK cell marker than DX5). Furthermore, if you want to use less kit on every spleen, think about passing your spleen suspension through nylon wool columns. It is a very cheap and effective way of getting rid of adherent cells, thus leaving you only to purify your NK cells from a pool of non-adherent cells, in which your NK cells are going to be? If you need more info let me know!
Good luck!
Has anyone tried a negative-selection approach? We have had good success designing our own purification kits for F480+ mono/macs and CD11c DCs. Might be worth trying if you are concerned about activation.
What is your cell sorter set up? While NK Cells might be pretty hardy, maybe you need a lower pressure/larger nozzle size. With a good clean prep, you should achieve >95% purity from a cell sorter. Sometimes you need a slightly broader sorting mask if you are losing too many cells. This can increase your yield without significant detriment to the purity.
The other important factors relating to activation are time and temperature.
Technically speaking getting high purity and high numbers is a contradiction. If you want to maximise your numbers you use Yield, but without good prep (including using the AutoMACS), purity suffers. You can use Purity 1/2 drop or 1 drop and get purity up around 99% but very low numbers. It comes down to a balancing act.
To achieve what you want you need to look at the whole procedure, and a mix of technologies. I like the idea of the nylon wool columns to remove adherent cells, never tried it, but it sounds good.
Do you guys know how many Nk cells we can get per spleen using the NK cell isolation kit from Miltenyi ? thanks
Hi Camila,
In fact, I used the NK cell kit on C57BL6/J mice and I could harvest between 800 000 to 1 million per spleen.
Good Luck