Nk macs media

Primary NK cells and apheresis cells were obtained from Senti Biosciences through overnight shipment. NK cells were isolated using the Human NK Cell Isolation Kit (Miltenyi Biotech, 130-092-657) via autoMACS system (Miltenyi Biotech). The feeder cells were prepared as described before^{20 (link)} by irradiation of the apheresis cells from the same donor with NK cells we used. In short, the apheresis cells were irradiated by MultiRad 350 (Precision X-ray) with 20 Gy under SnCuAI filter, and NK cells were mixed with the feeder cells at NK: feeder cells ratio of 1:5. The cell mixture was cultured in NK MACS media (Miltenyi Biotech, #130-114-429) supplemented with 5% human AB serum, 500 IU/mL of human IL-2, and 10 ng/mL of OKT-3 (Thermo Fisher, #14-0037-82). The population of NK cells and T cells were checked with anti-CD3-Alexa Fluor 647 antibody (1:100 dilution, Biolegend, #300322) and anti-CD56-APC-Cy7 antibody (1:100 dilution, Biolegend, #362511), and purified NK cells were maintained over 98% since day 7 of the co-culture.

Adhesion assay protocol was adapted from Strazza et al. [28 (link)]. Non-cancer control primary NK cells were isolated from PBMC by magnetic cell sorting using human NK cell isolation kit (Stemcell, Vancouver, British Columbia, Canada) according to manufacturer’s instructions. NK cells were resuspended at a density of 1 × 10⁵/100µL of NK MACS media (Miltenyi Biotec) supplemented with 100 IU IL-2 (Peprotech) and treated with 100 ng IL-15 (Immunotools) and/or 100 ng/mL fractalkine (Peprotech) for 2 and 24 h. NK cells were also treated with OAC patient-derived TCM or ACM for 2 and 24 h with and without pre-treatment with 5 nM CX3CR1 antagonist E6130 (MedChemExpress, Monmouth Junction, NJ, USA) for 1 h prior to exposure to ACM. A 96 well plate was coated with goat-anti-human IgG (Fc specific) in PBS and incubated overnight at 4 °C. Plates were subsequently coated with 2.5 µg/mL MAdCAM-1 (R&D, Minneapolis, MN, USA) for 1 h at 37 °C. NK cells were stained with CFSE and allowed to adhere for 20 min at 37 °C. Unbound cells were washed away. Unwashed wells were used as controls. The percentage of adherent cells was determined by a fluorescent plate reader and calculated as follows: $$\frac{\mathrm{Avg} \mathrm{intensity} \mathrm{in} \mathrm{well}}{\mathrm{Avg} \mathrm{intensity} \mathrm{in} \mathrm{unwashed} \mathrm{well}} \times \frac{100}{1}$$

PBMCs were supplied commercially (AllCells) and magnetically separated with NK Cell Isolation Kit (Miltenyi Biotec). Upon isolation, NK cells were cultured in complete NK medium, prepared with NK MACS media (Miltenyi Biotec) supplemented with human serum (1:20), penicillin streptomycin (1:100), NK supplement (1:100, Miltenyi Biotec), and IL-2 (500 IU/ml). Cells were transferred into a 24-well G-REX (Wilson Wolf) five days after isolation, and fresh NK media was then added every 2–3 days to ensure cell health. Purity was measured by flow cytometry using Miltenyi Biotec’s CD56 REAfinity (PE-Vio770; 1:50) at day nine of the NK cell culture. Cells were used for transfection 15–18 days after isolation.