Rpmi medium

Murine erythroleukemia cells (MEL-745A cl.DS19) were obtained from the German Collection of Microorganisms and Cell Cultures (Leibniz Institut, Braunschweig, Germany) and clone 19 is described to be DMSO sensitive, resulting in erythroid differentiation. Cells were grown in RPMI-medium (PAN BIOTECH, Aidenbach, Germany) containing 10% fetal calf serum (PAN BIOTECH) at 37 °C with 5% CO₂.

Using citrate phosphate dextrose adenine-containing tubes, we collected blood samples (30 mL) from three healthy dogs. The blood samples were diluted with an equal volume of phosphate-buffered saline (PBS) and then layered over Ficoll-Plaque PLUS (GE Healthcare Life Sciences, Little Chalfont, UK) in a conical tube. After centrifugation at 400 × g for 30 min, the buffy coat layer was carefully collected. The collected samples were incubated with red blood cell-lysis buffer at room temperature for 10 min. After adding PBS, each sample was centrifuged at 400 × g for 10 min, and the washing and centrifugation steps were repeated. Canine PBMCs (cPBMCs) were resuspended in Roswell Park Memorial Institute (RPMI) medium (Pan-Biotech, Dorset, Germany) supplemented with 10% EV-free FBS and 1% PS. Non-adherent cells were obtained after 24 hours.

For the potential references genes (ACTB, EEF2, GAPDH, NDUGB1, NDUFB4, PEA15 and PPA1), primers and probes were also self-designed (Biomers.net; Online Appendix Table 1). NDUFB4 and NDUFB1 are known to best meet published selection criteria for reference gene expression studies of human brain tissue [30 (link)]. Additionally, ACTB, EEF2, GAPDH, as well as PEA15 and EEF2 were selected from the online database for housekeeping genes and reference gene candidates (housekeeping.unicamp.br). Reference gene validation was performed using human astrocyte, microglia and microvascular brain endothelial cell lines (Applied Biological Materials, Richmond, Canada) kept in RPMI medium (PAN-Biotech, Aidenbach, Germany) and sampled at a minimum of four time points. Therefore, RNA was isolated using the RNeasy Kit (Qiagen), and reverse transcription was performed as described above. Genes were analyzed for their expression stability over time within each cell line as well as between the cell lines using NormFinder [31 (link)]. The reference gene should have the lowest possible intragroup variation (variation within samples of the same origin taken at different time points) as well as intergroup variation (variation within samples of different origin).

CD14 BD IMAG Beads (Becton-Dickinson, BD Austria) were used to isolate monocytes from the blood of normal healthy donors according to the manufacturer’s instructions. Written informed consent was obtained from all participating blood donors by the Central Institute for Blood Transfusion & Immunological Department, Innsbruck, Austria. The use of anonymized leftover specimens for scientific purposes was approved by the Ethics Committee of the Medical University of Innsbruck (EK1166/2018). Monocytes were re-suspended in RPMI medium (PAN Biotech, Germany) containing 5% AB serum and 2 mM l-glutamine and cultured with GMCSF (50 ng/mL) or MCSF (50 ng/mL) for 7 days at 37°C/5% CO₂. GMCSF-stimulated macrophages were further challenged with recombinant human IFNG (10ng/ml) and E. coli LPS (10 ng/mL) for 24h to obtain pro-inflammatory GMCSF^(IFNG/LPS) macrophages. MCSF-stimulated macrophages were challenged with recombinant human IL4 (10 ng/mL) and IL13 (10 ng/mL) to obtain anti-inflammatory MCSF^(IL4/IL13) macrophages. After stimulation, cells were washed with PBS three times to remove the activator cytokines and cultured in RPMI/2.5% FCS for an additional 24 h to generate conditioned media (CM). Finally, CM was harvested, centrifuged for 10 min at 300g, and filtered (pore size < 0.2μm) to eliminate any residual cellular components.

CT26 (mice colon adenocarcinoma syngeneic to BALB/c) was procured from ATCC. A549 (Human lung adenocarcinoma), Hela (Human cervical cancer), CHO (Chinese hamster ovary), NIH-3T3 (mouse embryonic fibroblast) cells were purchased from National Center for Cell Sciences (Pune, India). HFF (human foreskin fibroblast) cell was a kind gift from CCMB, Hyderabad. All the cells were checked to be mycoplasma free. CT26 cells were grown in Roswell park memorial institute (RPMI) medium (PAN Biotech, Aidenbach, Germany) while A549, Hela, HFF, CHO, and NIH-3T3 cells were expanded in Dulbecco’s modified Eagle’s medium (Sigma) both supplemented with 10% fetal bovine serum (FBS) (South American Origin, Lonza) and 1% penicillin–streptomycin–kanamycin at 37 °C in a humidified atmosphere of 5% CO₂ in air.