Team:TU-Delft/test4
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<h2><span lang=EN-US style='mso-ansi-language:EN-US'>Introduction:<o:p></o:p></span></h2> | <h2><span lang=EN-US style='mso-ansi-language:EN-US'>Introduction:<o:p></o:p></span></h2> | ||
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Flow | + | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Flow |
- | + | <b>cytometry</b> is used to measure the response of the mating | |
pathway when the receptor is induced by the ligand. Yeast goes into cell cycle | 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 | arrest (sticking in the G1 phase) after induction. After induction we take time | ||
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The DNA | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The DNA | ||
- | stain <span class=SpellE>Vybrant</span> < | + | stain <span class=SpellE>Vybrant</span> <b>DyeCycle</b> |
Green has excitation/emission peaks of 519/563 respectively. Filters of the <span | Green has excitation/emission peaks of 519/563 respectively. Filters of the <span | ||
class=SpellE>Cytek</span> Flow cytometer are 488 nm (Blue) and 561 nm (Yellow). | class=SpellE>Cytek</span> Flow cytometer are 488 nm (Blue) and 561 nm (Yellow). |
Revision as of 17:03, 23 September 2012
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.