Cellavista

Passage 5 GNV019 cells were plated at 15 cells/cm² on five PO-coated 10 cm dishes. 20 individual cells/dish were randomly selected on the subsequent day, marked with pen at bottom of dish, and followed for 30–60 days. Seven of these formed clonal colonies, were selected using 8 mm cloning cylinders (Corning), trypsinized, and transferred to a 6 cm dish for expansion. CL1/2/3/6/7 cells were depicted from these based on their distinctive morphologies. BN-samples were plated at 0.5 cells/well in up to eight 96-well plates, validated, and monitored throughout clonal expansion by automated image-based analysis (Cellavista, Roche). 11–26 single cell-derived subclones were selected per case and expanded. For generation of pilot data, at least 5 subclones were used per patient sample.

BV-2 female mouse microglia-like cells were grown in DMEM + 10% FBS, and THP-1 male human monocyte cells were grown in RPMI-40+ 10% FBS+ 0.05mM β-mercaptoethanol at 37°C, 5% CO₂. Adherent cells were split using 1X TrypLE (Gibco). For cellular assays, cells were treated with the following concentrations of drugs, unless otherwise noted, in DMEM (for BV-2) or RPMI-40 (for THP-1) without serum: 100-200μM ganciclovir, 10ng/ml IFNγ (R&D systems), 100ng/ml LPS (Sigma-Aldrich), 10μM Fludarabine (Selleckchem), 1μM Ruxolitinib (Selleckchem), 1μM TG-101348 (Selleckchem), 1μM Amlexanox (Tocris bioscience). Secreted signaling proteins were measured in conditioned culture supernatants from BV-2 cells stimulated with GCV for 24h in the absence of serum using two independent Luminex arrays (Human Immune Monitoring Center, Stanford University and Eve technologies, Canada). Nitrite assay was performed on conditioned culture supernatants of cells stimulated with drugs for 24h using the Griess Reagent System (Promega) according to manufacturer’s instructions. To assess cell viability, cell confluence was measured using an automated microscope (Cellavista; Roche). Toxicity was measured using Celltox Green cytotoxicity assay (Promega). All experiments were run in triplicates and replicated at least 3 times with cell lines and at least twice with primary microglia.