Mlc400b laser engine

Single colonies were picked and grown at 30 °C. Log-phase S. cerevisiae cells growing at 30 °C were transferred to 16 °C for 16 h. Cells were fixed with 75% ethanol for 1 h, sonicated for 5 s at 40% amplitude, and mounted in medium containing DAPI. Imaging was performed using an Apo TIRF 100 ×1.49 NA objective (Nikon, Plano Apo) on a Nikon Ti2 microscope with a Yokogawa-X1 spinning disk confocal system, MLC400B laser engine (Agilent), Prime 95B back-thinned sCMOS camera (Teledyne Photometrics), and a piezo Z-stage (Mad City Labs). The samples were blinded during imaging. The percentage of aberrant binucleate cells was calculated as the number of binucleate cells divided by the sum of wild-type and binucleate cells. Eight to 12 biological replicates from independent colonies were done for Figure 4c, and three were done for Figure 4d,i. At least 200 cells were counted for each biological replicate per condition.

Log-phase S. cerevisiae cells growing at 30°C were transferred to 16°C for 16 hr. Cells were fixed with 75% ethanol and stained with DAPI. Imaging was performed using a ×100 Apo TIRF NA 1.49 objective on a Nikon Ti2 microscope with a Yokogawa-X1 spinning disk confocal system, MLC400B laser engine (Agilent), Prime 95B back-thinned sCMOS camera (Teledyne Photometrics), and a piezo Z-stage (Mad City Labs). The percentage of aberrant binucleate cells was calculated as the number of binucleate cells divided by the sum of wild-type and binucleate cells. Six (Figures 3C—5), four (Figure 7A), or three (Figure 3D) biological replicates were performed from independent cultures for each condition.