Spinning disk microscope

We 3D-printed electrotaxis chambers (Figure 2—figure supplement 1) with dimensions of 20 mm × 5 mm × 0.25 mm and composed of a clear resin using a Formlabs Form2 3D-printer. Agar bridges were used to isolate cell media from electrodes to minimize electrochemical products and pH changes. Twenty V/cm constant EFs were applied. Time-lapse images of the phase-contrast channel and the RFP/GFP channel were recorded using PerkinElmer spinning-disk microscope at a frame rate of 0.1 frames/s (Yokogawa CSU-X1 spinning-disk scan head (5000 rpm)) with Hamamatsu EMCCD camera and Volocity analysis software.

C. albicans SC5314 that has the GFP gene integrated within the ENO1 genomic locus^{42 (link)}, was kindly provided by Prof. J. Berman (Tel Aviv University, Israel). The fungi were first seeded on potato-dextrose agar plates (Acumedia, Neogen, Lansing, MI) at room temperature where they grow in the yeast form, and then inoculated in RPMI (Sigma, St. Louis, MO) for a 16–18 h incubation at 37 °C to let them form hyphae. For the yeast-hypha transition assay, colonies of C. albicans in yeast form were inoculated in RPMI at an OD_600nm of 0.5 and incubated with different concentrations of AEA, AraS and 2-AG at 37 °C for 4 h. For time-lapse microscopy, hyphae that has been formed after 4 h incubation of C. albicans in yeast form (OD_600nm of 0.25) at 37 °C, were incubated in 300 μl RPMI in a μ-slide 8 well chambered coverslip (ibidi GmbH, Martinsried, Germany) in the absence or presence of 125 μg/ml AEA. The Okolab incubation chamber was used to maintain the temperature at 37 °C. Images were captured each 5 min for 3 h using the Nikon spinning disk microscope (Yokogawa W1), the × 20 CFI PLAN APO VC objective and the SCMOS ZYLA camera. The images were processed using the NIS-Element AR program.