Biotek synergy 2

Outer membrane permeability was determined according to the method by Wang et al. (Wang et al. 2021c (link)). Briefly, a 200 μL/well of bacterial suspension in TSB medium (pH 8.5, 3% NaCl) at mid-logarithmic growth phase (mid-LGP) at 37 °C was mixed with a 2 μL/well of 10 mm N-phenyl-1-naphthylamine (NPN) (Sangon, China). The change of fluorescence intensity per well was measured using BioTek Synergy 2 (BioTek, USA). The excitation and emission wavelengths were 350 nm and 420 nm, respectively (Wang et al. 2021c (link)). Inner membrane permeability was also determined (Huang et al. 2021 (link)). Briefly, a 200 μL/well of bacterial suspension was mixed with a 2.5 μL/well of 10 mM O-nitrophenyl-β-d-galactopyranoside (O-nitrophenyl)-β-d-galactopyranoside (ONPG) (Sangon, China). The mixture was incubated at 37 °C, and absorbance at OD_415 nm of each well was measured using BioTek Synergy 2 (BioTek, USA) every 30 min (Huang et al. 2021 (link)). Cell surface hydrophobicity and membrane fluidity assays were performed as described previously (Yang et al. 2020 (link)).

VRPs were produced by co-electroporation of in vitro transcribed YFV-Gluc replicon RNA and in vitro transcribed SIN-flavi-prME/C packaging construct RNA into BHK-J cells using the electroporation conditions as described [39 (link)]. After electroporation, cells were kept at 32 °C in MEM complete for 72 h. The VRPs were harvested by centrifugation of the cell supernatant at 1200 rpm for 10 min to remove cell debris. The cleared supernatants containing the VRPs were aliquoted and stored at −80 °C.
For the determination of VRP titers TCID₅₀ analysis was performed. For this, the VRPs were serially diluted in tenfold steps in MEM with four replicates per dilution. In 96-well plates, an equal amount of BHK-J cell suspension (2 × 10⁴ cells/well) was added to each dilution and incubated at 37 °C, 5% CO₂ for 48 h. Readout was performed via measurement of the Gluc activity using the Renilla Luciferase Assay System (Promega, Madison, WI, USA) and a plate luminometer (BioTek Synergy 2, Agilent, CA, USA). Wells with luciferase activity at least two-fold above the threshold measured for mock-infected cells were counted as positive. TCID₅₀ was calculated by the Spearman and Kärber algorithm as described [40 ] and converted to focus forming units (FFU)/mL with FFU approx. 0.69 × TCID₅₀.

CellTiter-Glo^® Luminescent Cell Viability Assay was used to measure the cell viability for transduced MJ and HH cells according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, MJ and HH cells transduced with INSL3 shRNAs or non-target shRNA lentiviral transduction particles were seeded at a density of 2000 cells per well in duplicate in white opaque 96-well microplates in 50 μL of medium. After incubation at 37 °C for 3 h, an equal volume of CellTiter-Glo^® reagent was added to each well. The cells were cultured for 24 h and/or 48 h, and the luminescence was measured by BioTek Synergy 2 plate reader (Agilent Technologies, Inc. Santa Clara, CA, USA).

To assess cell viability after bioprinting with each alginate bio-ink, a fragment of each printed construct was taken 1, 5, 7, 14, and 28 days after printing. The construct fragments were stained with live/dead viability/cytotoxicity kit for mammalian cells (catalog no. L3224, Invitrogen, Carlsbad, CA, USA) according to the standard protocol. After staining for 30 min, the construct fragments were placed on a concave glass microscope slide (catalog no. 7104, Chang Bioscience, Fremont, CA, USA) and imaged with a Leica DMi8 microscope (Leica Microsystems, Wetzlar, Hesse, Germany). Images were further analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). For each alginate at each time point, 6 samples were measured.
To assess cellular proliferation, proliferation assays were performed 1, 5, 7, 14, and 28 days after printing. At each time point, alginate constructs were moved to a new 12-well plate and submerged in 2 mL of fibroblast growth media. Next, 200 µL of Cell Counting Kit-8 (CCK8, catalog no. CK0411, Dojindo Molecular Technologies, Rockville, MD, USA) was added to each well. The constructs were incubated at 37 °C for 3 h. Using BioTek Synergy 2 (Agilent Technologies, Santa Clara, CA, USA) and BioTek Gen 5 Software (Agilent Technologies, Santa Clara, CA, USA), the optical density at 450 nm (OD₄₅₀) of each well was obtained.

We used a panel of 60 anti-cancer drugs (Table 2) to screen meningioma cells for drug sensitivity, as described previously [53 (link)]; the majority of these compounds are currently in clinical trials. Each compound was tested in four concentrations (10 μM, 1 μM, 100 nM, and 10 nM) in triplicate in a 384-well format. Early-passage cells (i.e., within the first five passages) cultured from PDOX tumors were plated at a density of 4000 cells/well and incubated at 37 °C with humidified 5% CO₂ for 7 days. Cell viability was measured with the BioTek Synergy 2 (Agilent, Santa Clara, CA, USA). IC50 (half maximal inhibitory concentration) values were determined by a nonlinear best-fit method using Excel Solver (Microsoft Office). We evaluated the effects of drugs on cell viability using the two-sample Kolmogorov–Smirnov test (KS2TEST) comparing each experimental well to the DMSO control. A dose-response curve was generated, and the normalized area under curve (AUC) was calculated by a nonlinear regression analysis using a four-parameter logistic equation (GraphPad Prism, version 5, GraphPad Software).

Aspartic protease activity from the cell lysate (protease inhibitors were not added to the lysis buffer) and from the cell culture media was measured by the hydrolysis of the fluorogenic 11-mer peptide substrate (7-methoxycoumarin-4-yl)acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH₂, (catalog no.: 219360; Merck-Calbiochem). Enhanced fluorescence after the cleavage of the Phe-Phe amide bond was spectrofluorimetrically determined at a pH of 4. Reaction mixtures contained 40 μl of 50 mM sodium acetate buffer, pH 4.0; 5 μl of 200 μM substrate solution; and 5 μl of sample (cell lysate or macrophage culture media). The reaction mixtures were incubated at 40 °C for 10 min, and the reactions were terminated with 200 μl of 10% trichloroacetic acid. The increase in fluorescence intensity produced by substrate cleavage during incubation was measured at an emission wavelength of 393 nm with excitation at 328 nm using a BioTek Synergy2 fluorescence spectrophotometer (Agilent Technologies). The specific enzyme activity was normalized to the protein concentration of the cell lysate or the cell culture media.