Live dead yeast viability kit

Cell viability was evaluated using a LIVE/DEAD^® Yeast Viability Kit (Thermo Fisher Scientific, Warsaw, Poland) according to the manufacturer’s instructions. After staining with a mixture of FUN^® 1 and Calcofluor^® White M2R, cells were inspected under an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. For each analysis, 200 cells were used.
Cell viability was evaluated for both ancestral strains and their evolved derivatives in: (a) YPD medium (1% w/v Difco Yeast Extract, 2% w/v Difco Yeast Bacto-Peptone, 2% w/v dextrose), (b) YPD medium supplemented with 1.5 M KCl, and (c) 30% YPGlu medium (1% w/v yeast extract, 2% w/v peptone, 30% w/v glucose). Cell viability was assessed at the logarithmic phase of growth that was previously established for each growth condition. Cell viability was compared between the ancestral strains and their evolved derivatives separately for each growth condition variant.

Calcofluor white M2R (CFW) and FUN1 from the LIVE/DEAD yeast viability kit (Thermo Fisher Scientific, Waltham, CA, USA) were used for staining to check the structure and viability, respectively, of hyphae on the glass coverslips at days 0, 1, 4, and 8 during incubation in the soil. SYBR green (Invitrogen Life Technologies, Carlsbad, CA, USA) staining was applied to check bacterial colonization of P. canescens hyphae after 8 days of incubation. The staining procedures are described in the supplemental material.
Microscopic observations of hyphae or bacteria stained with CFW, FUN1, or SYBR green were carried out using an Axioskop 2 fluorescence microscope (Zeiss, Oberkochen, Germany). A fluorescein isothiocyanate (FITC) filter (exciter filter band pass [BP], 450 to 490 nm; emission long pass [LP], 420 nm) was used to visualize fungi or bacteria stained by FUN1 and SYBR green, while a 4′,6-diamidino-2-phenylindole (DAPI) filter (exciter filter G, 365 nm; emission LP, 420 nm) was used to visualize CFW-stained hyphae or spores. All of the observations were performed using a 63× oil lens objective (total magnification, ×630; Zeiss). Images were captured using a Zeiss AxioCam digital camera and the AxioVision software.

Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MA, USA) (with MOPS, pH 7.2), dimethylsulfoxide (DMSO), Sabouraud dextrose agar (SDA; Difco), saline solution (SS), iMarKTM microplate reader (Bio-Rad, Hercules, CA, USA), FLC, Bioscreen C equipment, Bioscreen software (Growth Curves USA, Piscataway, NJ, USA), 100-well microplates (honeycomb), LIVE/DEAD™ Yeast Viability kit (Thermo Fisher Scientific, Waltham, MA, USA), Olympus FV 1000 confocal microscope, Tescan Lyra 3 microscope, Fmoc-Arg(Pbf)–OH, Fmoc-Trp(Boc)–OH, Fmoc-Gln(Trt)–OH, Fmoc-Leu-OH, Fmoc-Lys(Fmoc)–OH, Fmoc-6-Ahx-OH, Rink amide resin, dicyclohexilcarbodiimide (DCC), and 1-hydroxy-6-chlorobenzotriazole were purchased from AAPPTec (Louisville, KY, USA). Trifluoroacetic acid (TFA), acetonitrile (ACN), dichloromethane (DCM), N,N-dimethylformamide, ethanodithiol, triisopropylsilane, methanol, acetonitrile, and isopropanol were obtained from Merck (Darmstadt, Germany). SPE SupelcleanTM columns were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MA, USA) (with MOPS, pH 7.2), dimethylsulfoxide (DMSO), Sabouraud dextrose agar (SDA; Difco), saline solution (SS), iMarKTM microplate reader (Bio-Rad, Hercules, CA, USA), FLC, Bioscreen C equipment, Bioscreen software (Growth Curves USA, Piscataway, NJ, USA), 100-well microplates (honeycomb), LIVE/DEAD™ Yeast Viability kit (Thermo Fisher Scientific, Waltham, MA, USA), Olympus FV 1000 confocal microscope, Tescan Lyra 3 microscope, Fmoc-Arg(Pbf)–OH, Fmoc-Trp(Boc)–OH, Fmoc-Gln(Trt)–OH, Fmoc-Leu-OH, Fmoc-Lys(Fmoc)–OH, Fmoc-6-Ahx-OH, Rink amide resin, dicyclohexilcarbodiimide (DCC), and 1-hydroxy-6-chlorobenzotriazole were purchased from AAPPTec (Louisville, KY, USA). Trifluoroacetic acid (TFA), acetonitrile (ACN), dichloromethane (DCM), N,N-dimethylformamide, ethanodithiol, triisopropylsilane, methanol, acetonitrile, and isopropanol were obtained from Merck (Darmstadt, Germany). SPE SupelcleanTM columns were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Initially, the same procedure described above to determine the MIC and the checkerboard was performed, in the case of the combinations. After 48 h of incubation, the LIVE/DEAD™ Yeast Viability kit from Thermo Fisher Scientific was used and staining was performed according to the supplier’s instructions [61 ]. Briefly, the multiwell plate was centrifuged for 5 min at 10,000× g, the supernatant was discarded, and 80 µL of phosphate buffered saline (PBS), 20 µL of FUN™ 1, and 100 µL of Calcofluor™ White M2R (CW) were added, incubated for 30 min at 30 °C, and placed under an Olympus FV 1000 confocal microscope. Wavelengths used: 405, 532, and 488 nm.
This viability kit combines a two-color fluorescent probe for determining yeast viability, FUN™ 1, and a fluorescent reagent that binds to the cell wall surface, CW. Thus, if the integrity of the plasma membrane is preserved, the metabolic function of the yeast is observed when it converts the yellow-green-fluorescent intracellular staining of FUN™ 1 into red-orange intravacuolar structures. CW labels chitin with blue fluorescence regardless of metabolic status [61 ].

Yeast cells from xylan mono- and co-cultures were sampled (100 μL) at different time points (24, 48, 72, and 96 h), centrifuged (10,000 × g, 5 min), and washed in 1 mL 10 mM HEPES buffer, pH 7.2. Co-cultures were stained with 0.15 μL FUN-1 and 0.5 μL calcofluor white in 100 μL 10 mM HEPES buffer, pH 7.2, plus 2% glucose using a LIVE/DEAD yeast viability kit (Thermo Fisher, Germany) for 30 min in the dark at RT. Fluorescence images were recorded using a Leica DFC360 FX microscope (Germany) with a 100×oil immersion objective and a DFC 360 FX camera. Excitation filters for yellow fluorescent protein (YFP; 480 to 520 nm) were used for FUN-1 using an exposure time of 75 ms with 1.8 gain, and an excitation filter A4 (320 to 400 nm) was used for calcofluor white using an exposure time of 5 ms and 1.8 gain. Emission spectra were designated yellow and blue for FUN-1 and calcofluor white, respectively, in LAS X (Leica) software. Total fluorescent particle area and pixel intensity values in the 12-bit images (1,392 by 1,040 pixels) were used to differentiate and quantify blue and yellow fluorescence using ImageJ (Fiji) software.

The effect of sphingosine on the cell wall of Candida was determined by calcofluor white staining, as previously described [57 (link),58 (link),59 (link)], with a few modifications. N. glabrataa DSM70614 was cultivated in YPD broth at 35 °C up to the mid-exponential phase. We treated 2 to 5 × 10⁵ cells per mL for 4 h (37 °C, 200 rpm) with two separate concentrations of sphingosine at a final concentration equal to 1 × MIC or 2 × MIC. After incubation, the cells were harvested, washed with 2% D-(+)-glucose (Sigma-Aldrich) containing 10 mM Na-HEPES (Sigma-Aldrich), pH 7.2, and adjusted to a concentration of 5 × 10⁷ cells/mL. Candida incubated in 70% ethanol for 4 h was used as a positive control [60 (link)].
The samples were stained for viability with LIVE/DEAD Yeast Viability Kit (ThermoFisher Scientific, Waltham, MA, USA) containing two fluorescent probes (FUN 1) and cell wall staining (calcofluor white M2R), according to the manufacturer’s instructions. Concisely, 100 µL of the yeast suspension containing 10 μM FUN1 was mixed with 100 μL calcofluor white M2R (final concentration, 25 μM). The cells were incubated for 30 min at 30 °C in the dark and were analyzed by fluorescence microscopy.