Stayrna solution

34 pairs of matched glioma tissues and blood samples were collected during standard neurosurgical tumor removals at the Department of Neurosurgery, Institute of Psychiatry and Neurology (Warsaw, Poland). Additionally, five independent random controls of blood samples were obtained from healthy volunteers. All solid tissues were submerged in the stayRNA solution (A&A Biotechnology) and stored at –80°C. Blood was collected into EDTA-treated tubes and centrifuged at 3500 rpm (MPW, Centrifuge MPW-350R, Rotor 1236B) for 10 min at 4°C. Then supernatant (plasma) was immediately transferred into the clean Eppendorf tubes and stored at –80°C.

PBMCs were isolated from 9 ml samples of whole blood using LYMPHOSEP™ (MP Biomedicals LLC, Ohio, USA), according to the manufacturer's instructions. The obtained pellet was suspended in 4 ml of a Lymphogrow medium (Cytogen-Polska Sp. z o.o., Zgierz, Poland) containing phytohemagglutinin (PHA) and recombinant IL-2 (4 ng, 100 U, BioLegend, San Diego, CA, USA). The suspension was then transferred to a 25-ml vessel for adherent culture. Cells were grown under standard conditions at 37°C, 5% CO₂ with shaking for 24 h. Non-adherent cells were washed with PBS and transferred to a 25-ml vessel for suspension culture with fresh Lymphogrow medium supplemented with IL-2. After another 48 h, the cells were passaged and maintained in a culture using a standard RPMI-1630 medium supplemented with L-glutamine (2 mM), FBS (10%), penicillin (100 IU/ml), streptomycin (100 μg/ml), and with the addition of IL-2. Cell differentiation was measured by CD3, CD4, CD8, CD45, and HLA-DR by flow cytometry analysis. In the third passage, anti-TNF mAbs' (Sigma) was added (10 μg/ml). In parallel, a control culture without the addition of the antibody was carried out. After 72 h of culture, cells were suspended in 200 μl stayRNA solution (A&A Biotechnology, Gdansk, Poland) and frozen at −80°C for RNA isolation. Moreover, part of the cells was subjected to apoptosis analysis.