Isolation and Cryopreservation of Immune Cells from Ovarian Cancer Samples

9 ml of peripheral blood were harvested into heparinized tubes (Sarstedt, Germany). Venous blood specimens were gathered before the operation procedure of OC patients. Fresh ascites fluid was collected aseptically and small pieces of neoplastically changed primary tumour tissue (~ 1 cm³) from nonmargin areas and without necrotic changes were obtained during the surgical resection. Blood and ascites specimens were centrifuged (1500 rpm/10 min) to obtain rendered cell-free fluids and the supernatants were immediately stored at − 80 °C for later analysis. OC patients’ blood plasma and ascites fluid were used immediately after defrosting and were not subjected to further freeze–thaw cycles. For isolation of tumour-infiltrating immune cells, freshly resected tumour tissue was minced with scissors into 2- to 4-mm, and placed into a gentleMACS C tube containing 5 ml of dissociation medium. Next, all tumour specimens were processed using Tumor Dissociation Kit (Miltenyi Biotec, Germany) to obtain single-cell suspension, following manufacturer’s instructions. The resulting cell suspension was filtered through 70-mm mesh filter (BD Biosciences, USA). Mononuclear cells (MCs) were obtained from blood, ascites and tumour tissue by gradient separation as we previously described in details [2 (link)] and were cryopreserved until use.