335
12. Perform steps 4–7 to induce the mating pathway, but do not
add cycloheximide.
13. Transfer 2 mL of each culture to an empty 5 mL round bottom
tube with a 35 μm cell-strainer cap. Dispense the liquid culture
through this cap to further reduce aggregation.
14. Run the filtered cultures in a cell sorter:
15. Vortex a 5 mL round bottom tube containing 500 μL of sterile
drop-out medium and place it in the cell sorter’s collection
chamber.
16. Set up the gating strategy outlined in Fig. 2a to target live, single
cells. Most cell types (including bacteria, yeast, and animal cells)
will form cell aggregates when suspended in solution. Thus,
there is a risk that a simplistic gating strategy (e.g., selecting cells
that display high GFP fluorescence) will be compromised by the
presence of cell aggregates that may include cells with low fluorescence levels considered to be a single “event” by the flow
cytometer. To minimize this risk, it is important to devise a multigate selection strategy that includes at least two gates based on
forward and side scattering signals capable of distinguishing
between single cells and aggregates. In the case of yeast cells, two
gates are sufficient: one plotting the width of the forward scattering signal vs. the height of the forward scattering signal, and a
second gate plotting the width of the side scattering signal vs. the
height of the side scattering signal, as shown in Fig. 2a. Similar
strategies can be devised for other cells types, though it is important to be familiar with the specific aggregation propensities and
light scattering properties of your cell of choice.
17. Use your negative control to establish the cells’ baseline fluorescence and your positive control to visualize the fluorescence of
active mutants. Draw a gate that includes the latter but does not
overlap with the former. Note that in separate experiments you
may want to select cells that activate the pathway in the absence
of stimulus, or only in the presence of stimulus (Fig. 2b).
18. Sort the desired number of mutants and collect them in a 5 mL
tube containing 500 μL of drop-out medium. Once the sort has
ended, lightly vortex the collection tube to wash the walls.
19. Plate the content of the collection tube onto solid medium
(100 μL per plate). Approximately 40% of the collected events
can be expected to be recovered this way.
Fig. 2 (continued) and side scattering heights). (b) Gating strategies needed to select cells that activate the
pathway response upon pheromone treatment. Note the exclusion of inactive mutants (left). (c) Flowchart of a
cell sorting experiment. A plasmid library of STE2 mutants is transformed in yeast. An initial phenotypic screen
shows that most mutants cannot sense the pheromone of K. lactis as well as wild type. Following a first round
of cell sorting, almost all mutants selected can sense the pheromone and about half can do so better than wild
type. A second cell-sorting step yields an even greater proportion of strong K. lactis-responsive mutants
Rewiring Signaling Networks
12. Perform steps 4–7 to induce the mating pathway, but do not
add cycloheximide.
13. Transfer 2 mL of each culture to an empty 5 mL round bottom
tube with a 35 μm cell-strainer cap. Dispense the liquid culture
through this cap to further reduce aggregation.
14. Run the filtered cultures in a cell sorter:
15. Vortex a 5 mL round bottom tube containing 500 μL of sterile
drop-out medium and place it in the cell sorter’s collection
chamber.
16. Set up the gating strategy outlined in Fig. 2a to target live, single
cells. Most cell types (including bacteria, yeast, and animal cells)
will form cell aggregates when suspended in solution. Thus,
there is a risk that a simplistic gating strategy (e.g., selecting cells
that display high GFP fluorescence) will be compromised by the
presence of cell aggregates that may include cells with low fluorescence levels considered to be a single “event” by the flow
cytometer. To minimize this risk, it is important to devise a multigate selection strategy that includes at least two gates based on
forward and side scattering signals capable of distinguishing
between single cells and aggregates. In the case of yeast cells, two
gates are sufficient: one plotting the width of the forward scattering signal vs. the height of the forward scattering signal, and a
second gate plotting the width of the side scattering signal vs. the
height of the side scattering signal, as shown in Fig. 2a. Similar
strategies can be devised for other cells types, though it is important to be familiar with the specific aggregation propensities and
light scattering properties of your cell of choice.
17. Use your negative control to establish the cells’ baseline fluorescence and your positive control to visualize the fluorescence of
active mutants. Draw a gate that includes the latter but does not
overlap with the former. Note that in separate experiments you
may want to select cells that activate the pathway in the absence
of stimulus, or only in the presence of stimulus (Fig. 2b).
18. Sort the desired number of mutants and collect them in a 5 mL
tube containing 500 μL of drop-out medium. Once the sort has
ended, lightly vortex the collection tube to wash the walls.
19. Plate the content of the collection tube onto solid medium
(100 μL per plate). Approximately 40% of the collected events
can be expected to be recovered this way.
Fig. 2 (continued) and side scattering heights). (b) Gating strategies needed to select cells that activate the
pathway response upon pheromone treatment. Note the exclusion of inactive mutants (left). (c) Flowchart of a
cell sorting experiment. A plasmid library of STE2 mutants is transformed in yeast. An initial phenotypic screen
shows that most mutants cannot sense the pheromone of K. lactis as well as wild type. Following a first round
of cell sorting, almost all mutants selected can sense the pheromone and about half can do so better than wild
type. A second cell-sorting step yields an even greater proportion of strong K. lactis-responsive mutants
Rewiring Signaling Networks