Replica plater for 96 well plate

To express Ycf1 and mutants for the cadmium susceptibility assay, S. cerevisiae strain BY4742 with endogenous Ycf1 knockout (Horizon Discovery) were transformed following the Frozen-EZ Yeast Transformation II protocol (Zymo Research). Transformed yeast strains were grown for 48 hours on YNB-His agar plates at 30 °C. Individual colonies were picked and diluted to approximately 0.2 OD₆₀₀ using sterile ACS grade water (Midland Scientific) that was further filtered with a 0.22 μm syringe filter. Cells were then spotted onto YRG (yeast nitrogen base with ammonium sulfate 0.67% w/v, raffinose 1% w/v, galactose 2% w/v, CSM-His 0.077% w/v and 2% w/v agar) agar plates with and without 100 μM CdCl₂ using a replica plater for 96 well plate (Sigma-Aldrich). Four biological replicates of each experimental condition were performed. Images were collected following 5 days of incubation at 30 °C with a Bio-Rad Chemidoc MP Imaging System (Bio-Rad) and analyzed using the ImageJ software (50 (link)).

Measurements of cell density were performed by measuring absorbance at 600 nm using a Gensys 6 UV-Vis spectrophotometer (Thermo Fisher). Measurements of cell density and cell size were performed using a Coulter Z2 Particle Count and Size Analyzer (Beckman Coulter) with a 100- $\mathrm{\mu }$ m aperature. For comparative growth assays, cells were spotted onto relevant solid growth media. Cell spotting was performed by dilution of a stationary phase culture to an initial OD₆₀₀ of 1.0, followed by 10-fold serial dilutions. All dilutions were then spotted onto solid media using a Replica Plater for 96-well Plate, either the 8 × 6 or 12 × 8 array as needed (Sigma-Aldrich). Plates were incubated at indicated temperatures for indicated times as noted in the figures and legends. At least 3 independent biological replicates were performed on different days for spotting assays shown in figures, and a representative image is shown.

Measurements of cell density were performed by measuring absorbance at 600 nm using a Gensys 6 UV-Vis spectrophotometer (Thermo Fisher). Measurements of cell density and cell size were performed using a Coulter Z2 Particle Count and Size Analyzer (Beckman Coulter) with a 100- $\mathrm{\mu }$ m aperature. For comparative growth assays, cells were spotted onto relevant solid growth media. Cell spotting was performed by dilution of a stationary phase culture to an initial OD₆₀₀ of 1.0, followed by 10-fold serial dilutions. All dilutions were then spotted onto solid media using a Replica Plater for 96-well Plate, either the 8 × 6 or 12 × 8 array as needed (Sigma-Aldrich). Plates were incubated at indicated temperatures for indicated times as noted in the figures and legends. At least 3 independent biological replicates were performed on different days for spotting assays shown in figures, and a representative image is shown.