Abstract:

 

Introduction:

Flow cytometry is used to measure the response of the mating pathway when the receptor is induced by the ligand. Yeast goes into cell cycle arrest (sticking in the G1 phase) after induction. After induction we take time points where we stain the yeast DNA and use a flow cytometer to measure fluorescence intensity and correlate it with DNA amount.

We calibrate the system by inducing yeast strains with alpha pheromone and measuring excitation peaks and distribution and compare it with non-induced strains. The side scatter is a parameter which is influenced by the cell morphology.

The DNA stain Vybrant DyeCycle Green has excitation/emission peaks of 519/563 respectively. Filters of the Cytek Flow cytometer are 488 nm (Blue) and 561 nm (Yellow). We consider peaks obtained by blue excitation.

Figure 1 Fluorescence excitation and emission spectra for the Vybrant® DyeCycle™ Orange stain bound to DNA in TBE,pH 8.3. Source: http://www.invitrogen.com/site/us/en/home/References/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-cycle-analysis/vybrant-dyecycle-green-and-orange-stains.html

Methods:

A frozen stock is grown overnight on 30 °C and diluted until an OD₆₀₀ of approximately 0.05 was measured. Cells were grown in DO –Leucine media. Cells transformed with nicotinic acid receptor where grown in DO –Leucine –Nicotinic acid media. Cells where centrifuged (10 min 4000 RPM) and media was refreshed. Cells were grown for seven hours and centrifuged again. Pellet size is estimated and evened. Media was refreshed again. After 2.5 hours of growth (cells in exponential phase) cells were induced with ligand. Half an hour before every time point 1 ml cells is taken and 2 μl Vybrant® DyeCycle™ Orange is added, vortexed and kept on 37 °C for half an hour and then measured with a Cytek FACScan. Graphs were analyzed with Flowjo.

Results

We take S. cerevisae with I7-OR1G1 expression as an example, Time points are taken at ~T=1 hour, ~T=3.20 hours and T=4.30 hours after induction with α pheromone. Figure 2 shows graphs of side scatter versus intensity and a histogram of cell intensity of the whole population (N=20.000 cells).

Positive control

A secondary cloud can be seen to emerge in the pheromone induced population which show a reduced side scatter (lower cloud centre). The higher intensity indicates an increased amount of DNA.

Figure 2 Cell intensity distribution of alpha pheromone induced S. cerevisae cells. The cells have been transformed with I7-Olfr154. Every time point shows an intensity distribution (top) of (Blue 590-20) excited cells. The lower graph shows a SSC versus intensity distribution.

 

Induction of Methyl nicotinate receptor

Figure 3 shows results cells transformed with I7-Gpr109A were induced by concentrations of methyl nicotinate. The negative control, non-induced cells over time, show two clouds relatively close to each other, similar to the negative control of the alpha induced cells. Cells induced with methyl nicotinate show very low alteration of intensity, only at t=3.25 hours a slight cloud shift towards the 10³ intensity level can be observed. Cells induced with nicotinic acid show a very broad shift from an intensity of 10⁴ to an intensity of 10³. This indicates that the DNA content per cell dropped. The specific negative control without receptor but with a high nicotinic acid receptor also has a drop in DNA content. This indicates that a high nicotinic acid can be the cause of the drop of DNA content, for example through increased cell division. Therefore, vitamin addition

Figure 3 Cell intensity distribution of ligand induced S. cerevisae cells. The cells have been transformed with I7-Gpr109A. Every time point shows an intensity distribution (top) of (Blue 590-20) excited cells. The lower graph shows a SSC versus intensity distribution.

 

Induction of Banana smell (isoamyl acetate) receptor

Figure 4 shows results of cells transformed with I7-Olfr154, I7-OR1G1 and wildtype cells were induced by concentrations of isoamyl acetate. The negative control, non-induced cells over time (first and fourth column), show two clouds relatively close to each other, similar to the negative control of the alpha induced cells. The same holds for the wildtype cells (third column). The I7-Olfr154 transformed cells (second column) shows at T=1.20 a slight increase in cell intensity, combined with a low SSC. At time points t=3.08 and t=4.30 this cannot be observed and the data shows little deviation from the negative control. The I7-OR1G1 transformed cells however, maintain this deviation in all three time points. There can also be noticed that this deviation is towards the 10⁵ region instead of 10³ with the nicotinic acid induced cells, similar to the secondary cloud of the alpha pheromone induced cells.

Figure 4 Cell intensity distribution of ligand induced S. cerevisae cells. The cells have been transformed with I7-Olfr154 and I7-OR1G1. Every time point shows an intensity distribution (top) of (Blue 590-20) excited cells. The lower graph shows a SSC versus intensity distribution.

 

Induction of diacetyl receptor

Figure 3 shows results of cells transformed with I7-Odr10 were induced by concentrations of 2,3 butadione (diacetyl). The negative control, non-induced cells over time show a deviation towards the 10³ region. The gain has been lowered between T=1.06 and T=3.18 points since high intensity points ‘dropped off’. No significant difference between the induced and non-induced data can be seen. What can be noted is that no secondary region can be detected, which can be seen for wiltype induced with diacetyl.