Ficoll paque plus density centrifugation

Monocytes were isolated from the venous blood of healthy volunteers. All participants gave written informed consent prior to the study in accordance with the 2013 Declaration of Helsinki. The study was approved by the local ethics committee of the Pirogov Russian National Research Medical University (Moscow, Russia; protocol #169 from 20.11.2017). Mononuclear cells were isolated by Ficoll-Paque Plus density centrifugation (GE Healthcare, Pittsburgh, PA, USA). Mononuclear cells were resuspended in complete RPMI 1640 medium (supplemented with 10% FBS, 1× GlutaMAX, and 1× penicillin–streptomycin solution; all from RBL Life Technologies, Waltham, MA, USA), plated (1 × 10⁶/ml) in six-well plates, and incubated at 37°C (5% CO₂). After 90 min, nonadherent cells were removed. To generate monocyte-derived macrophages (MDMs), 100 ng/ml of granulocyte–macrophage colony–stimulating factor (GM-CSF, SCI-store, Russia) was added to the isolated monocytes, and cells were cultured for 6 days.

Human immune cells were isolated from venous blood of healthy volunteers and patients with malignant melanoma. Specimens were collected with informed written consent in accordance with the Declaration of Helsinki. The study was conducted at King’s College London, King’s College London, Guy’s and St Thomas’ NHS Foundation Trust (08/H0804/139 approved by London Bridge NRES committee; 16/LO/0366 approved by London-Central NRES Committee). Human peripheral blood mononuclear cells (PBMC) were isolated from 40 ml blood using Ficoll^® Paque Plus density centrifugation (GE Healthcare).