NK cells magnetically isolated from peripheral blood mononuclear cells (PBMC) can have very small proportions of myeloid cells contaminating the isolation. These are very sensitive to contaminants and can greatly enhance NK cell functional responses if present (http://www.ncbi.nlm.nih.gov/pubmed/24337374). For this reason it is best to use highly purified ('LEAF' or 'NA/LE') antibodies for triggering in these studies. In addition to the antibodies against specific human receptors to be investigated, this assay should include non-specific control antibodies (an isotype control antibody) and surface-bound non-activating antibodes (e.g. anti-CD56) to see the background levels of functional response when NK cells form contacts with P815, but activating receptors are not triggered. Receptor-independent activation (e.g. PMA + ionomycin) can serve as a useful positive control for degranulation and cytokine production.
Antibody concentrations should be titrated to find a dose that induces robust functional responses when NK cells are co-incubated with P815, but not when then are incubated with the antibody alone. The antibodies listed were all titrated to 1 microgram/ml for the current assay.
1. Pre-incubating the NK cells with antibodies, rather than pre-incubating the P815 cells with antibodies
The binding of the specific anti-human receptor antibodies is generally stronger to the human receptor than it is to the mouse Fc receptor, so the original protocol had the pre-incubation step with NK cells. However, our assays involve a single target cell line, but multiple NK cell conditions that take a significant amount of time to prepare. Given the duration of the assay, for time saving reasons, we changed the incubation step to be with the P815 so we could start the incubation while the NK cells were being prepared. Preliminary optimization assays showed similar results were obtained by this method as to when the antibodies were first pre-incubated with the NK cells.
2. Washing off antibodies after a pre-incubation step
Some published methods wash antibodies away after the initial pre-incubation step on NK cells. A comparison of these two variations (washing or not washing) prior to addition of P815 showed a titrable response from the NK cells in both cases. The washing step greatly increased the amount of antibody that was required to get maximal responses without obviously providing any other benefit. For this reason the wash step was removed from our assay.