Dal1100

In this study, IR exposures were performed by using a Cs¹³⁷ gamma ray source at NASA Ames Research Center and accelerated particle beams at the NSRL at Brookhaven National Laboratory in New York. WT and rad51Δ cells dried in 10% trehalose in Stripwell microplates or inside microfluidic cards were exposed in both a desiccated state and liquid suspension. When exposed in liquid suspension, cells were rehydrated in Synthetic Complete (SC) growth medium containing 1 × aB metabolic indicator dye (DAL1100; Invitrogen) before each exposure (Santa Maria et al., 2020 (link)).

For osteoblast differentiation, primary COBs were cultured in an osteogenic medium containing 0.1 mg/mL ascorbic acid (Sigma-Aldrich, A8960, Saint Louis, MO, USA), and 10 mM β-glycerophosphate (Sigma, G9422, USA), and incubated with Alamar blue solution (Invitrogen, DAL1100, Carlsbad, CA, USA; Dilution 1:10) for cell proliferation. Subsequently, cells were washed and incubated with a solution containing 6.5 mM Na₂CO₃, 18.5 mM NaHCO₃, 2 mM MgCl₂, and phosphatase substrate (Sigma-Aldrich, S0942 St. Louis, MO, USA), and alkaline phosphatase activity was measured by an iMark microplate absorbance reader (Bio-Rad, 1681130, Hercules, CA, USA). To assess extracellular matrix mineralization in mature osteoblasts, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Fixed cells were washed twice with distilled water and stained with a 2% Alizarin red solution (Sigma-Aldrich, A5533, St. Louis, MO, USA) for 10–20 min. Cells were then washed three times with distilled water and examined for the presence of calcium deposits.

Strains were grown in duplicate from a total of 10⁴ spores for 48 h in a 96-well plate in a total volume of 200 µl minimal medium (MM) per well. MM was supplemented with either xylose or glucose, the specified concentration of 2-deoxy-glucose (2DG), and 10% (vol/vol) alamarBlue (Invitrogen DAL1100). The absorbance at 570 nm and 600 nm of 50 µl of the grown culture supernatant was read using a SpectraMax I3 platform (Molecular Devices). Growth curves are based on the calculated 570-nm/600-nm ratio and subtraction from the negative controls (containing no spores). Positive controls were processed without 2DG and presented 100% of growth of the respective strains.

Advantage was taken of the Alamar Blue viability assay ^{101 (link)}. Briefly, wells of deep 96-well plates (1896–2000, USA Scientific) each containing 0.8 ml of LB broth with 50 μg/ml of kanamycin (BP906–5, Fisher Scientific) were singly innoculated with 10 μl aliquots of each of 3,884 knockout strains for 16 hours at 37°C on a rotator (160 rpm). 200 µl of a 1:1000 dilution of each (~10⁶ cfu/ml) in 1 mM Na₂HPO₄, 0.18 mM KH₂PO₄, 0.27 mM KCl, 13.7 mM NaCl, pH 7.2 containing 10% Alamar Blue (DAL1100, Invitrogen) in wells of black 96-well plates were monitored hourly for five hours in a SpectraMax M3 plate reader (Molecular Devices; San Jose, CA) with 3 s agitation before reading simultaneously from the well bottoms at medium PMT voltage with a 590 nm cutoff filter and six flashes per read for the Alamar Blue fluorescence signal (excitation/detection = 560/590 nm). The same hourly monitoring continued for 30 hours after the addition of N-104, N-80/C-25 (negative control), or ampicillin (positive control; BP1760–5; Fisher Scientific) at a final concentration each of 100 µM. Data for each of 3,884 strains were normalized to untreated with 10% Alamar Blue background subtracted as the ratio of the viability slope during the first five hours of N-104 treatment versus that five hours previous.

Seeded scaffolds (n = 3 per scaffold group in culture medium with/without osteogenic supplement) were transferred to fresh 24-well plates and 1 mL of alamarBlue™ (DAL1100, Invitrogen, Paisley, UK) working solution, (diluted 10× from stock solution with phenol-free Dulbecco’s modified Eagles medium (DMEM, 11880, Gibco, Paisley, UK) supplemented with 10% FBS and 1% antibiotics) was added per well, and samples were incubated at 37 °C with 5% CO₂ for 3 h, after which each well content was transferred to a cuvette and absorbance measured at 570 nm in a uv/vis spectrophotometer (Spectronic Camspec Ltd., Garforth, UK). Absorbance at 600 nm of phenol-free DMEM was read and subtracted from the test sample absorbance to obtain the final value.