Cell Cycle Synchronization of Schizosaccharomyces pombe by Centrifugal Elutriation of Small Cells

1. At least 20 h before elutriation, dilute the 400-mL preculture (5 × 10⁶ cells/mL) in EMM2 in 5-L flasks to produce 7 L of culture at 3.8 × 10⁶ cells/mL on the day of elutriation. Do not exceed 1.8 L per flask. Grow the culture with rotation at 240 rpm or faster for at least 20 h (until Step 7).

• Culture conditions are critical. If cell density exceeds 3.8 × 10⁶ cells/mL, synchrony will be compromised in the second cycle; this suggests that cells from a higher culture density are more prone to starvation in the separation chamber. If density is far below 3.8 × 10⁶ cells/mL, the reduction in the robustness of the gradient will compromise size selection. Similar issues apply to culture volume, speed, and late inoculation.

2. Secure the assembled rotor in the centrifuge with an anchor cable, clamp the culture supply and efflux tubes to the cable, and check that the tubes will remain attached to the rotor when the anchor cable is taut. Assemble the other components according to Figure 1A. Set the centrifuge temperature to match the culture temperature.

• If elution is to be performed at 36°C, store the assembled rotor overnight in an incubator at 36°C.

3. Label 31 microcentrifuge tubes from 0 to 500 in increments of 20. Aliquot 50 µL of 30% formalin into each tube before closing the lids.

4. Label 50-mL centrifuge tubes for sample collection in Step 11.

• Cell mass increases exponentially; hence, the volume required to maintain biomass will decrease exponentially, and so the volume sampled at each point throughout the culture should decrease exponentially (i.e., if 50 mL is collected at t = 0 at 25°C, then 37.5, 21, 15.75, and 11.8 mL will produce the same yield at each 100-min interval).

5. On the day of elutriation, place a bottle of fresh EMM2 in the water bath at the culture temperature. Pump prewarmed EMM2 through the system at 160 mL/min with the exhaust tube from the rotor returning to the same bottle. Remove the bubble trap from its clamp and invert so the inlet is uppermost until the trap is 60% full of medium, and then reinvert and clamp with inlet below (leaving a large air pocket in the bubble trap).

6. To purge the rotor of bubbles, set the rotor speed to 2000 rpm and press Start. If the “back pressure” reaches 8 psi, press stop immediately and check the connections. Stop the centrifuge as soon as the speed reaches 2000 rpm.

• Small bubbles will emerge when the speed returns to 130–100 rpm.

7. Begin cell loading as follows.

• i. Set the rotor speed to 4000 rpm while maintaining the pump rate at 160 mL/min. At 4000 rpm, adjust the stroboscope to see the chamber.

• ii. Place a culture flask from Step 1 next to the bottle of medium in the water bath.

• iii. If eluting at 36°C, run prewarmed medium through the rotor for 20 min before loading cells.

• iv. Switch the input from fresh medium to the culture flask. Move the tube for the exhaust medium from the rotor to a waste vessel.

  • The chamber will become increasingly cloudy until enough cells accumulate to condense into a dense mass in the lower half of the chamber. A clear “front” will separate the mass of cells from the clearer void above it (Fig. 1B, left).

8. Once the front has formed, increase the pump rate to 196 mL/min.

• The front will move toward the top of the chamber.

9. Continue loading as follows.

• i. Periodically check the position of the front and the back pressure. Maintain a small gap between the front and the top of the chamber by slowing the pump rate. (Adjust the strobe to maintain sight of the chamber.) Reduce the pump rate if the back pressure exceeds 5 psi.

  • The front will continuously move toward the top of the chamber as shown in Figure 1B, center.

  • Wild-type cells can tolerate back pressures up to 8 psi; however, the synchrony of mutants can be compromised above 5 psi.

  • See Troubleshooting.

• ii. Periodically replenish the culture from the additional culture flasks and aerate by shaking the feeder culture. If the air pocket in the bubble trap shrinks below 10%, introduce more air by repeatedly lifting the intake tube from the culture.

  • The air pocket in the bubble trap acts as a shock absorber to dampen surges created by the pump.

10. When the medium exiting the rotor is clear (usually 20 min after starting the run), collect 200 mL of this conditioned medium in a bottle and place it in the water bath.

11. Twenty-seven minutes after initiating loading, begin elution as follows.

• After 27 min, the front between the void space and the dark mass of cells will be very stable. If cells spend >27 min in the chamber, the synchrony in the second cycle will be compromised, suggesting that cells begin to starve after 27 min.

• i. Switch the input from the culture to a shaken bottle of fresh medium and decrease the centrifuge speed by 50 rpm increments until the profile matches that in Figure 1B, right. Tape the exhaust tube across the top of a 500-mL bottle (to keep its height constant throughout elution as in Fig. 1A).

  • The level of synchrony will drop if the height of the collecting tube is not fixed throughout the sampling.

• ii. Begin collecting 50-mL samples in 50-mL centrifuge tubes. Check the cells in each sample by microscopy.

  • Elution of cells can be detected with a spectrophotometer at OD₅₉₅ = 0.05–0.06; however, microscopy has the advantage of immediate indication of size selection.

• iii. If cells have not begun to elute after 50 mL have been collected, reduce the centrifuge speed by further increments of 50 rpm until they appear.

  • The void volume in the system is 50 mL or slightly more, so always collect 50 mL before making the next change.

12. Once cells are eluting, add 150 mL of the conditioned medium from Step 10 to the reservoir of fresh medium. Continue to collect samples in 50-mL tubes throughout elution, checking the size selection and transferring all good samples to a 5-L flask. Because the physical properties of the gradient change as cells leave, periodically reduce the centrifuge speed by 50-rpm increments to maintain elution. Continue replenishing the reservoir with fresh medium.

• Addition of conditioned medium guards against the possibility that S. pombe will use small molecules for intercellular communication.

13. Once the sampling is complete, count the cell number and dilute with medium to 1 × 10⁶ cells/mL. Return the cells to the shaking incubator. For >1.8 L, use two flasks with an identical volume in each. Do not grow >1.8 L of eluted culture in a 5-L flask.

• The ratio of the culture volume to flask volume sets the precise periodicity of the cell cycle of the size-selected culture.

14. Every 20 min, remove 500 µL from the culture and transfer it into the appropriately labeled 1.5-mL tube containing formalin for calcofluor staining to monitor septation index as described in Protocol: Chromatin and Cell Wall Staining of Schizosaccharomyces pombe (Hagan 2016). (If using two flasks, take consecutive samples from each flask.)

• Remember to accommodate the exponential increase in biomass (see Step 4) when taking specific samples.

• Septation index can also be monitored using a phase contrast microscope by creating a “pseudodark field” illumination. With a 40× phase 2 objective, the phase ring should be moved on the condenser from the phase 2 toward the phase 3 setting until the field of view becomes black, yet the cell outlines and septa shine brightly.

• See Troubleshooting.