Epithelial cell growth supplement

HaCaT cells, a human keratinocyte epithelial cell line (America Type Culture Collection) were cultured under standard conditions as reported^{15 (link)}. The primary human choroid plexus epithelial cells (HCPEpiCs) (ScienCell Research Laboratories) were cultured in EpicM epithelial cell medium added with 1% epithelial cell growth supplement (ScienCell Research Laboratories) and 2% fetal bovine serum (FBS). HCPEpiCs cells were used within three cell-culture passages to exclude de-differentiation to mesenchymal cells.
Chemical reagents were from Sigma, if not specified. Human plasma purified Cp was from Enzo Life Sciences. The antibodies used were: anti-ceruloplasmin (ab8813, Abcam); the antibodies used for the signaling analysis (FAK #3285, p-Tyr³⁹⁷FAK #3283, ERK1/2 #9102, p-Thr^202/Tyr²⁰⁴ERK1/2 #9101, GSK3β #9315, p-Ser⁹GSK3β #9336, AKT #4691, p-Ser⁴⁷³AKT #193H12) were from Cell Signaling Technology, while anti-β-tubulin antibody (T6199) was from Sigma-Aldrich.

Human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA, USA) were grown in an epithelial cell medium (ScienCell, Carlsbad, CA, USA) containing epithelial cell growth supplement (ScienCell, Carlsbad, CA, USA), in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. HRPTEpiC was used at passages 8–10. Cells were plated at a density of 3 × 10⁵ cells/well in 6-well plates and incubated for 3 days. Fresh medium containing doxorubicin (DOXO) (Sigma, St. Louis, MO, USA) was added to cells and cultured for 24-h to induce cellular senescence. Similarly, HRPTEpiC was treated with fresh medium containing H₂O₂ (Sigma, St. Louis, MO, USA). Cells were harvested at the end of the treatment for further analysis.

Human primary choroid plexus epithelial cells (HCPEpiC, ScienCell™ Research Laboratories, Carlsbad, CA, USA) were cultured in Epithelial Cell Medium (ScienCell) containing 2% fetal bovine serum (FBS, ScienCell), 1% Epithelial Cell Growth Supplement (ScienCell), and 1% Penicillin/Streptomycin solution (ScienCell) at 37 °C and 5% CO₂. For culturing these cells, the manufacturer’s protocol was followed. Cell density was set to 6000 cells per cm² and poly-L-lysine (ScienCell) -coated (2 µg/cm²) BioLite cell culture flasks (Thermo Scientific, Rochester, NY, USA) were used.

Primary human HSCs and primary human intrahepatic biliary epithelial cells (HiBECs, cholangiocytes) were purchased from ScienCell Research Laboratories (5300 & 4100, respectively). HSCs were cultured in stellate cell medium (ScienCell, 5301) supplemented with 2% fetal bovine serum (FBS), stellate cell growth supplements, and 1% penicillin/streptomycin (P/S). HiBECs were cultured in epithelial cell medium (ScienCell, 4101) supplemented with 2% FBS, epithelial cell growth supplement (ScienCell), and 1% P/S. Cells were grown in flasks precoated with poly-l-lysine and subcultured with trypsin/EDTA and trypsin neutralizing solution (ScienCell, 0103 & 0113, respectively) according to supplier protocol. LX2 cells (Merck, catalog SCC064) were cultured in DMEM, with 10% FCS. In cell culture experiments, supernatants were assayed for CCL24 using ELISA (R&D Systems, catalog DY343).

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.

Recombinant SARS-CoV-2 (Wuhan variant) spike protein subunits S1 and S2, as well as the RBD of S1, were purchased from Raybiotech (Peachtree Corners, GA, USA). Human alveolar epithelial cells (HPAEPiC), epithelial cell medium and epithelial cell growth supplement were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Mouse and human recombinant macrophage colony-stimulating factor (M-CSF) were obtained from PeproTech (Rocky Hill, NJ, USA). BD Vacutainer CPT tubes were purchased from Fisher Scientific (Waltham, MA, USA). RBC lysis buffer, penicillin–streptomycin, thioglycollate medium FITC-dextran, TRITC-dextran, and H₂DCFDA were purchased from ThermoFisher (Waltham, MA, USA). RPMI-160 media was purchased from Cytiva (Marlborough, MA, USA). Fetal bovine serum was purchased from GeminiBio (West Sacramento, CA, USA). Calphostin c, LY294002, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), diphenyleneiodonium chloride (DPI), polymyxin b, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). GSK2795039 was purchased from Tocris Biosceince (Minneapolis, MN, USA).