Biolite 6 well plates

HCPEpiCs (2 × 10⁵/well) were then treated in BioLite 6-well plates (Thermo Scientific, Rochester, NY, USA) with metHb, ferrylHb, heme (25–50 µM), or recombinant TNF-α (100 ng/mL, Gibco, Carlsbad, CA, USA) as control for 24 h to replicate IVH-induced inflammatory cellular conditions in vitro. MetHb and ferrylHb were prepared from fresh peripheral blood samples obtained from healthy volunteers, as we have previously described [27 (link)]. After treatment, supernatants were collected and stored at −80 °C before RNA isolation and measurement of protein biomarkers. To analyze the degree of cell death caused by treatments, LDH leakage was determined in the supernatants of stimulated HCPEpiC cell cultures via photometric measurement of catalytic LDH activity on a Cobas^® 6000 analyzer (Roche Diagnostics, Mannheim, Germany).

Primary human choroid plexus epithelial cells (HCPEpiC, ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in a special Epithelial Cell Medium (ScienCell) containing 2% fetal bovine serum (FBS, ScienCell), 1% Epithelial Cell Growth Supplement (ScienCell), and 1% Penicillin/Streptomycin solution (ScienCell) in a 5% CO₂ humidified atmosphere (95%) at 37 °C. For culturing these cells, we followed the manufacturer’s protocol. Cell density was set to 5000 cells per cm² and poly-L-lysine (ScienCell) coated (2 µg/cm²) BioLite cell culture flasks (Thermo Scientific, Rochester, NY, USA) were used. HCPEpiC cells (2 × 10⁵/well) at passages 4–6 were subcultured into BioLite 6-well plates (Thermo Scientific) and then treated with ex vivo IVH-III, IVH-IV, or non-IVH control CSF samples (10 v/v %) for 24 h. After treatment, cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS) solution (Lonza, Walkersville, MD, USA), then lysed in 1 mL TRI Reagent (Molecular Research Center, Cincinatti, OH, USA) and stored at −20 °C before RNA isolation.