For preparing NKEM media:
IL-2 can be added to the NKEM stock at the start of the experiment or added fresh as needed. We see no difference in expansion rate or viability.
General advice for refreshing media in G-Rex wells:
Carefully remove media from the top of the wells. Do not disturb the layer of cells at the bottom of the wells. If cell layer disturbance occurs, rest G-Rex in the incubator again for a couple hours before attempting to remove media.
General advice for harvesting cells from G-Rex:
Carefully remove 60mL media from the top of the wells, then resuspend the cell layer with leftover 40mL media in the well. Transfer the resuspended cells into 50-mL conical tubes. This allows you to fit one well of cells into one 50-mL conical tube.
Spot check each batch of frozen cells by thawing at least one vial and recording (1) the yield, (2) the viability straight after thaw and (3) the viability the day after thaw. Keeping an eye on these values ensures reproducibility of the cell product.
General advice for preparing and irradiating feeder cells:
For convenience and reproducibility, we irradiate feeder cells in large batches then store them as frozen aliquots until needed. We generally use freshly processed trimacones to start these expansions, but the assay should work with thawed cells. If working with thawed PBMC, rest the cells overnight (4x106/mL in R10; 5% CO2, 37°C) then proceed with NK cell enrichment.
Let feeders grow for at least one week post thaw before irradiating. Grow feeders in R10, splitting down to 1x105/mL on Mondays and Wednesdays, and down to 5x104/mL on Fridays. Our irradiator (BioRad) can work with tubes or flasks, but maximally fits 7x 50mL tubes at 1x107/mL. Seal the tube caps with parafilm. After irradiating, place cells on ice, return to the lab and freeze down the cells (using the cell counts obtained prior to irradiation). We recommend aliquots of 10-50x106cells/mL in 90% FBS, 10% DMSO. There is some loss upon thaw - be sure to wash cells and recount before adding them to NK cells.