Crystal violet

Biological interleukin-1 activity was determined as described previously^{40 (link)}. Briefly, human skin fibroblasts (5000 cells/well; 100 µl/well) were cultured overnight in 96-well plates (Nunc, Thermo Fisher Scientific). The medium was aspirated and replaced by fresh medium (Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose (Biochrom), containing 1% l-glutamine, 1% antibiotics, and 10% fetal calf serum (Thermo Fisher Scientific, Schwerte, Germany). Serial 1:4 dilutions of standard (recombinant IL-1α, 10 ng/ml; PeproTech, Hamburg, Germany) or samples were performed and cultured for 96 h. The cell layers were washed, fixed with 3% formaldehyde (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), and stained with crystal violet (Serva Electrophoresis GmbH, Heidelberg, Germany). The absorption was read at 540 nm in an ELISA reader (Spectrafluor, Tecan, Crailsheim, Germany), the data plotted and the ED₅₀ (median effective dose) determined. The biological activity (U/ml) was calculated with respect to the recombinant standard (10 U/ml ≙ 10 ng/ml recombinant IL-1α).

For performing plaque reduction assays, frozen spleen, heart and lung tissues were used for preparation by homogenizing in 1 mL DMEM using a Precellys 24 homogenizer (Bertin Technologies, Montigny le Bretonneux, France). MEF cells were seeded at 1.5 × 10⁵ cells/well in 12-well cell culture plates and infected on the following day utilizing dilutions of organ homogenates. After 90 min of adsorption, DMEM supplemented with 0.3% agarose (Serva, Heidelberg, Germany) was added. agarose was removed 5–7 d p.i., depending on plaque formation. Cell layer was fixed and stained with 1% crystal violet (Serva, Heidelberg, Germany) in 20% ethanol solution, followed by three washing steps using PBS, drying and counting of plaques with an inverted light microscope.

To determine the long-term effect of IR on the cell survival, the colony-forming units assay was performed. Therefore, pADSCs (1000 cells), MCF-7 (500 cells), MCF10A (1000 cells), and ZR-75-1 cells cells (8000 cells) were seeded in triplicates in T₂₅ flasks or 6-well plates (Greiner Bio One) and treated with the radiation doses ranging from 0 Gy to 6 Gy. A full medium exchange with culture medium was performed every three (ADSCs) or seven days (MCF-7, MCF10A, ZR-75-1). On day 7 (MCF10A), day 21 (ZR-75-1 cells), day 15 (MCF-7 cells), or on day 20 (pADSCs), the cells were stained by 1% crystal violet (Serva Electrophoresis GmbH, Heidelberg, Germany) to visualize formed colonies. Colonies consisting of at least 50 cells were counted by microscopy. The calculated plating efficiency (PE) and survival fractions (SF) were evaluated using the data analysis and graphics software Origin 8.6.

Drug interactions in anti-MCMV efficacy were assessed in MEFs in a GFP-based approach similar to the HFF/HCMV setting using MCMV-GFP. Deviations were the number of seeded cells (1.5 × 10⁵ cells/well), as well as the time period in between infection and harvest (5 days). For assessing drug efficacy against the recombinant MCMV-UL97, an in vitro infection PRA was performed in MEFs. Four 12-well culture plates were seeded with MEFs at 1.5 × 10⁵ cells/well and infected one day later with MCMV-UL97. After virus adsorption of 90 min, the inoculum was replaced by medium supplemented with single compound, compound combination or solvent control, as well as 0.3% agarose (Serva, Heidelberg, Germany). A serial dilution of 6 concentrations/single compound starting with app. 4 × EC₅₀ and a serial dilution of 8 concentrations of the combination, starting with the highest concentrations of both single dilution series, were used. All infections were performed in biological duplicates. agarose was removed 5 d p.i., the cell layer was fixed and stained with 1% crystal violet (Serva, Heidelberg, Germany) in 20% ethanol solution, and plaques were counted under the microscope. Antiviral efficacy (mean of biological duplicates) was expressed as percentage of solvent control and entered into CompuSyn software (Version 1.0 [59 ]; ComboSyn, Inc., Paramus, NJ, USA).

MNCs of each BSM type were cultivated in 6-well plates with 1 × 10⁶, 4 × 10⁶ and 10 × 10⁶ cells per well as duplicates at a final cell density of 1 × 10⁵, 4 × 10⁵ and 10 × 10⁵ MNC/cm². Medium was changed every 3rd day. At day 14, cells were washed with PBS and incubated in 0.5% (w/v) crystal violet (SERVA Electrophoresis, Heidelberg, Germany) in 20% (v/v) methanol (Merck, Darmstadt, Germany) for 30 min at RT followed by rinsing with aqua dest. Colonies were defined as circular arrangements of cells of more than 50 stained cells, indicating that one viable cell as colony forming unit gave rise to a colony through replication [13 (link),62 (link)].