We have tried to purify NK-cells using MACS-columns and the NK-cell untouch kit from Miltenyi. This works pretty well, however, if there are still some contaminating T-cells (even
Hi Marleen,
you could also try MACS CD3 depletion followed by CD56 enrichment. This is done in the clinics to avoid contaminating T cells and works pretty good.
What kind of feeder cells do you use?
Good luck with your experiments,
Markus
Hi Markus,
We use theMiltenyi NK untouch kit, and this works pretty good already (with
Hi, Marleen.. Is there particular reason to use IL-2? One of the reasones T cells grow faster could be IL-2 as they expressed higer receptor(s), a, b, g. It is well konwn that IL-15 is important at the early stage of NK cell differentiation although T cells will also responde to IL-15 as well (perhaps less than IL-2). Otherwise you should use sorter (FACSAria).. I feel that you have a reason to use untouch KIT (even though the sorting gives a high purity).. Good luck ..
It's best to start with freshly isolated PBMCs. I would stain with CD3 and CD56, facs sort at low pressure (15psi) to avoid shearing of the NK cells and then plate into pre-prepared feeder cell cultures (we normally use a 1: 1 mix of irradiated (40 Greys) Daudi cells and RPMI 8866 cells) with human serum (male AB-, 10%) and IL-2 (50U/ml). For plating the NK cells we normally use a ratio of 1 feeder cell per 3 NK cells. Leave for 5-7 days and then stain for CD3 and CD56. You can modify the cocktail of antibodies if it is of interest to sort particular subpopulations of NK cells.