Trisodium citrate blood collection tubes

Healthy donor blood collected in trisodium citrate blood collection tubes (Sarstedt) diluted with Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco, Life Technologies Limited) in a 1:1 ratio and overlayed on a 1.077 g/mL separation medium (Biocoll, Merck Millipore). Density centrifugation was performed at room temperature (400 g for 30 min) without acceleration and brake. Same methodology was used during the isolation of T cells from patient ascites samples. PBMCs were collected and plastic adherence was performed to deplete monocytes by incubating them in a T175 flask (Sarstedt) at 37 °C and 5% CO₂ for 1 h. For NK isolation, NK MACS Isolation Kit (Miltenyi Biotec) was used according to the manufacturer’s instructions. After isolation 5 U/ml recombinant human IL-15 (50 µg, Immuno Tools) was added to NK cells that were used for functional experiments after incubation overnight. For T cell isolation, CD3 human microbeads (Miltenyi Biotec) were used according to the manufacturer’s instructions. Purity of isolated T cells was routinely tested. In case of overnight resting 5–10 U/ml of recombinant human IL-2 (50 µg, Immuno Tools) was added to T cells.

Healthy donor blood was collected in trisodium citrate blood collection tubes (Sarstedt). Blood was diluted (1:1 ratio) with Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco, Life Technologies Limited) and overlaid on a separation medium (1.077 g/mL, Biocoll, Merck Millipore). Separation by density was done by centrifugation at room temperature (400g for 30 min) without acceleration or brake. Collected PBMCs were incubated in a T175 plastic flask (Sarstedt) at 37°C and 5% CO₂ for 1 h for depleting monocytes. NK isolation was performed using NK MACS Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. After isolation, 5 U/mL recombinant human IL-15 (50 µg, Immuno Tools) was added to NK cells that were used for functional experiments after incubation overnight.