Figure 1

Flash freezing and semi-automated precision grinding of fission yeast in liquid nitrogen. (a) Pellet of fission yeast cells (beige) in a centrifuge bottle. The cells were harvested by centrifugation, washed and pooled in water, then re-pelleted and drained. (b) Pellet resuspended using minimal volume of INCA buffer (see text), then transferred to tip-capped syringe. (c) Syringe inserted into tube adapter (gray) and centrifuged in horizontal swinging-bucket rotor. Arrow indicates horizontal rotation. (d) After centrifugation, the total (upper boundary of light-beige supernatant) and pellet (upper boundary of dark-beige cell pellet) volumes were recorded by direct reading from the syringe scale. (e) Supernatant removed and plunger re-installed. (f) Tip cap removed, and packed fission yeast swiftly extruded into liquid nitrogen (blue) as a continuous bead or "noodle". (g) Noodle broken into 3- to 7-mm pieces in liquid nitrogen. (h) Pieces loaded into pre-cooled (liquid nitrogen) motorized precision mortar grinder and ground in liquid nitrogen at the minimum pressure setting ("0"). Arrows indicate directions of rotation of the mortar and contents (white arrows) and pestle (black arrows). (i) When slurry of ground material appears homogenous, pressure setting is raised to pre-determined optimum ("0/1" in our case) until release of intact nuclei, as determined by fluorescence microscopy, is maximized. (j) Cryo-ground cells transferred to storage container for evaporation of liquid nitrogen at -80°C. Octagons denote steps at which samples may be safely stored at -80°C (initially only loosely capped to allow venting of the evaporating nitrogen). White-bordered octagons: -80°C storage optional; solid red octagon: storage required. (k) 200-μl pipette tip bevel-cut and bent for collecting samples of mortar contents for microscopy during grinding