Xenolight d luciferin k salt bioluminescent substrate

GAP43 transgenic mice were anesthetized with 2% isoflurane in 100% oxygen in an induction chamber, and injected intrapretioneally (i.p.) with different luciferase substrates on consecutive days: VivoGlo (VivoGlo Caspase 3/7 Substrate; Z-DEVD-Aminoluciferin Sodium Salt, Promega, Madison, WI, USA) followed by luciferin (XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate, PerkinElmer, Waltham, MA, USA), 24 hours after^{4 (link),13 (link)}. Animals were individually placed in the heated light-tight imaging chamber of IVIS SPECTRUM Imaging System (PerkinElmer, Waltham, MA, USA) with continuous inhalation anaesthesia of 2% isoflurane–oxygen mixture at 1 L/min. To obtain the baseline values, imaging was done 3 days before the surgery and then 3, 7, 14, and 28 days after tMCAO. Total flux of photons was measured using the Living Image 4.3 acquisition and imaging software (PerkinElmer, Waltham, MA, USA), as described previously^{13 (link)}.

Optical in vivo imaging was performed using the Xenogen IVIS Spectrum (Caliper Life Sciences Inc., MA, USA). Bioluminescent images were acquired 9 min after intraperitoneal injection of luciferin (150 mg per kg body weight, XenoLight D-Luciferin - K⁺ Salt Bioluminescent Substrate, PerkinElmer, MA, USA). During imaging, mice were anesthetized with 2% isoflurane gas in the oxygen flow (1.5 L per min). Signal intensities were quantified and analyzed using Living Image Software version 4.5 (PerkinElmer, MA, USA).

After euthanasia, the peritonea, spleens, livers, and diaphragms of the mice were removed. These organs were dissected and placed in 96-well white plates. The peritoneal lavage fluid (150 μl) was also placed in white plates. After the addition of luciferin (150 μg/ml, XenoLight D-Luciferin-K + Salt Bioluminescent Substrate, PerkinElmer), organ luminescence was measured using the EnVision™ Multilabel Plate Reader (PerkinElmer).

All bioluminescent S. aureus strains were grown in TSB overnight at 37 °C in a shaking incubator at 240 rpm. Bacterial cells were harvested by centrifugation (5000 rpm for 10 minutes), washed twice in the same volume of phosphate buffered saline (PBS) and resuspended in TSB. Chloramphenicol (10 μg/mL) was added to cultures when needed to maintain plasmid stability. Absorbance at 600 nm (A₆₀₀) was adjusted to 0.05 for all strains. 200 µL of bacterial culture were added to black 96-well plates with clear bottoms (Corning) and plates were incubated at 37 °C with shaking in a humidified microtiter plate shaker (Stuart). A Tecan Infinite M Plex plate reader was used to periodically measure bacterial growth (A₆₀₀) and luminescence intensity (Integration time 1,000 milliseconds). For Fig. S1A, values from quadruplicate wells were averaged and luminescence was expressed as relative luminescence units corrected to normalized bacterial growth (A₆₀₀). The experiment was repeated three times with biological replicates. For Fig. S2, values from triplicate wells without or with addition of different concentrations (0.125–5 mg/ml) of D-Luciferin (XenoLight D-Luciferin - K⁺ Salt Bioluminescent Substrate, PerkinElmer) were averaged and luminescence was expressed as relative luminescence units (RLU). The experiment was repeated three times with biological replicates.

The animals inoculated with Luc⁺/HCT116 and treated as described above were also imaged for the expression of luciferase to follow their tumor growth. For the optical imaging, mice were subcutaneously injected with the XenoLight D-Luciferin - K+ salt bioluminescent substrate (PerkinElmer, UK) at a dose of 10 µL/g of body weight before the luciferase detection. Mice were anesthetized and placed in the dark chamber of a IVIS^® LUMINA XMRS optical imaging systems (PerkinElmer, Waltham, MA, USA) for whole-body animal imaging and the emitted photons were quantified and analyzed using Living Image^® Software (PerkinElmer, Waltham, MA, USA). Imaging of live animals was performed twice a week.

AH4807 (lux), AH4775 (luc), AH4826 (lux + luc), USA300 LAC::lux, LAC4303 (lux) and Xen36 (lux) (all 1 × 10⁴ CFU) were cultured in 96-well black polystyrene microplates with clear bottoms that were tissue culture (TC)-treated (Corning) without (none) and with the addition of different concentrations (0.03–1.2 mg/240 µL/well) of D-Luciferin (XenoLight D-Luciferin - K⁺ Salt Bioluminescent Substrate, PerkinElmer) for 0–14 hours at 240 rpm at 37 °C. Absorbance (A₆₀₀) and bioluminescent signals (photons/0.1 second) were measured every 5 minutes on a plate reader (BioTek H1 Synergy) (n = 4 replicates with 2 iterations). All absorbance and bioluminescent signal measurements were blanked to control wells containing the same D-Luciferin concentration.