Lsrii multi laser analyzer with hts

Cells with appropriate omega-zinc finger and multi-reporter plasmids were cultured until ‘cloudy' (OD600 0.1 and above but not saturated). Cells were pelleted and resuspended in NM. Cells were expanded for 1 h at 37 °C. Cells were pelleted and washed four times in minimal media that lacks histidine, uracil and IPTG. Cells were resuspended in 1 ml of this media, titred in 10-fold dilutions on rich plates with the appropriate antibiotics, and stored at 4 °C overnight. On the basis of the overnight titre results, a volume of the culture held at 4 ^oC that contains 10 million cells was used to start a 15-ml culture of minimal media containing 100 μM IPTG and various inhibitors as indicated (6-aza (2 pg ml⁻¹), 5-FOA (2 mM), and/or 3-AT (5 mM)). These cultures were grown from 16–24 h but not allowed to reach OD600 above 0.8. Cells were recovered and resuspended in PBS plus 0.1% Tween. The mean fluorescence of each sample was measured with a BD LSRII Multi-Laser Analyzer with HTS (BD Biosciences, Sparks, MD, USA). Mean fluorescence values were determined from at least 20,000 cells. Each zinc-finger–reporter pair was assayed in triplicate.

We created diploid GEV strains that carried both an inducible HOG gene under the control of P _GAL1 promoter and a HOG pathway transcriptional reporter (P _STL1-yEVenus) to assay for downstream transcriptional activation in response to overexpression of various HOG pathway proteins. Strains were grown with agitation in low fluorescence media at 30°C to mid-log (Klett 80), at which point 200 μl of cell culture was sampled for flow cytometry by adding it to 800 μl of cold PBS + 0.1 % Tween 20 stored at 4°C. Each culture was induced by adding β-estradiol to a final concentration of 10 μM. Cultures were sampled for flow cytometry after induction with β-estradiol at T=2 hours and T=19 hours. Fluorescence was analyzed by flow cytometry on a BD LSRII Multi-Laser Analyzer with HTS (BD Biosciences).