Luciferin ef

To confirm the bioluminescence emission capabilities of red-shifted bioluminescent strains, serial 4-fold dilutions of fungal inoculums (1×10⁷ initial spore count) resuspended in 200 µl of PBS and D-luciferin (final concentration 0.15 µg/ml, Luciferin-EF, Promega, USA) were transferred to a 96-well plate (Nunclon Delta-surface, Thermo Fisher Scientific, Denmark); a well without conidia (PBS+luciferin) was used as a control. BLI signal in samples was measured using an IVIS Spectrum imaging system (PerkinElmer, USA). Consecutive images were acquired for 10 min using an exposure time of 30 s (open filter, F/stop 1, subject height of 0.5 cm and medium binning). Peak total flux was quantified using Living Image Software (version 4.5.4) from a circular region of interest (ROI) of 0.8 cm diameter covering each well. Red-emission spectra (total flux) characteristics of the red-shifted luciferase were determined using a range of emission filters from 520 to 740 nm, and compared to the BL Af2/7/1 strain (green spectrum). The LOD of ungerminated conidia of each bioluminescent strain was determined as the last significant measurement (total flux) above background levels of the serial 4-fold dilutions of fungal inoculums (1×10⁷ initial spore count) compared to control well measurements.

Triplicate 10-fold serial dilutions of the harvested fungal suspension were prepared in a black 96-well plate (Nunc^® Microwell, Thermo Fisher Scientific, Merelbeke, Belgium). An equal volume of D-luciferin solution (final concentration 0.15 µg/ml in PBS; Luciferin-EF, Promega, USA) was added. Images were acquired using an IVIS Spectrum system (Perkin Elmer, Waltham, USA) with the Living Image software (version 4.5.2) and an exposure time of 1 min, F/stop of 1 and medium binning. Individual regions of interest (ROIs) were placed over each well for quantification of the total photon flux. Afterwards, the number of CFUs per well was determined by triplicate plating of serial dilutions on Sabouraud agar, cultured as specified above.
For analysis of the emission spectra from bioluminescent C. neoformans strains, bioluminescence of the fungal suspensions was measured using emission filters from 520 to 800 nm (step size 20 nm) and an exposure time of 30 s. The observed total photon flux per emission filter was expressed relative to the total photon flux in the filter ‘open’ setting and a cubic spline curve was fitted.

HEK 293 cells expressing the GloSensor (Promega, Madison, WI, USA) and CXCR4 were cultured in a black 96‐well plate (Perkin Elmer, Waltham, MA, USA) for 2–3 days until the cells were confluent. Cells were then incubated with a HBSS/20 mM HEPES/2.5% Luciferin‐EF (Promega) solution for 2 h. Luminescence was determined using a plate reader (infinite F2000PRO; Tecan, Männedorf, Switzerland) until steady state was achieved. Forskolin (1 μM) was added 28 min after the stimulus, and the luminescence was recorded.