Dulbecco s modified eagles medium glutamax

HFF-1 cells (ATCC-SCRC-1041, ATCC) were cultivated in Dulbecco’s Modified Eagles Medium Glutamax (Thermo Fisher Scientific), supplemented with 5% FBS superior (Merck) and 5 μl of 10⁶ units/ml recombinant human FGF (PeproTech) / 500 ml Medium. HCMV-pp150-EGFP-gM-mCherry was a kind gift by Christian Sinzger [61 (link)]. The HCMV-TB40-BAC4 was a kind gift by Wolfram Brune [23 (link)]. HCMV-Merlin-pAL1502-WT and HCMV-Merlin-pAL1502-pp150-EGFP-gM-mCherry were a kind gift by Christian Sinzger [74 (link)]. For infection experiments, we used cell-free, infectious supernatant derived from HFFF-TetR cells that was a kind gift of Christian Sinzger. In these cells, RL13 and UL128 expression is suppressed as described by Murrell et al. 2017 [73 (link)] such that virions used for infection were lacking RL13 and UL128. Imaging was done on normal HFF-1 without the tet-repressor such that the analyzed cells expressed RL13 and UL128 [24 (link),73 (link)].
Different multiplicities of infection (MOIs) were used for the infection experiments. In general, low MOI infections were used to avoid artifacts generated by high virus doses. Therefore, whenever possible, we used MOIs between 0.5 and 1. However, for particular experiments, such as bulk assays or electron microscopy, we used MOIs of up to 5. The used MOI is indicated for each experiment.

Functional activity of ASK2131 at the OXT receptor was assessed using a Ca²⁺ flux fluorometric imaging plate reader assay. In a typical experimental protocol, stably transfected Chinese hamster ovary (CHO) hOTR NFAT bLac cells were plated at a density of 10,000 cells/well in a 384-well plate in growth medium (Dulbecco’s modified Eagle’s medium + Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), 10% dialyzed fetal bovine serum). The next day calcium-sensitive, fluo-4 dye (Invitrogen, Carlsbad, CA, USA) and probenecid (Invitrogen; 2.5 mM) was dissolved in 20 mL warm dye loading buffer (Hanks’ balanced salt solution with calcium and magnesium, 20 mM Hepes, pH 7.4) and added to each well. Plates were incubated for 60–120 min in a 37 °C cell incubator to allow cells to uptake the dye. The control peptide (OXT) and ASK2131 were diluted in Hanks’ balanced salt solution with calcium and magnesium, 20 mM HEPES, and 0.1% bovine serum albumin. After dye-loading, the cell plates were placed on the fluorometric imaging plate reader. A total of 15 mL of test compound was added to the 30 mL of dye on the cells, and intracellular Ca²⁺ was monitored for 90 s. Intracellular Ca²⁺ concentrations were calculated as the maximum increase over basal levels. Responses were normalized to control responses. Data were analyzed by curve fitting.

On postnatal day 2, rat pups were sacrificed and the cortex was isolated by dissection. The hippocampus and meninges were removed and the cortical gray matter was dissociated in PBS-glucose 0.2%. Astrocytes were then separated from other cells using a Percoll 30% gradient (GE Healthcare, Chicago, IL, USA). Cells were finally washed in PBS-glucose and seeded in gelatin-coated 175 cm² flasks. Astrocytes were left to proliferate at 37 °C in a humidified atmosphere containing 5% CO₂ in Dulbecco’s modified Eagle’s medium (glutaMAX, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 50 mg/mL penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 50 mg/mL fungizone (Thermo Fisher Scientific, Waltham, MA, USA) for two weeks. The medium was renewed after one week. On day 15, cells were trypsinized and seeded into multi-well plates for 2 days in medium supplemented with 10% of FBS. On day 17, the serum concentration was decreased to 3% and, when indicated, the medium was supplemented with the G5 supplement, a growth factor cocktail (Thermo Fisher Scientific, Waltham, MA, USA).