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Jump To: Page 1 Page 2 Page 3 Page 4 Page 5 Paint-A-Gate Tutorial
Purpose:
·
Evaluate phagocytosis in Tetrahymena pyriformis
·
Introduce the principles of
fluorescence microscopy and flow cytometry: data acquisition (log FSC vs log
SSC; and log SSC vs FL-1) and analysis (Paint-A-Gate PRO).
Student Preparation:
·
Microbiology lecture: Students should
be familiar with distinguishing cellular characteristics of Teterahymena sp and Saccharomyces sp. in comparison to other microbes. The techniques of light microscopy,
electron microscopy, fluorescence microscopy and flow cytometry should be
familiar.
·
Microbiology Laboratory: Students will
have learned techniques of microbial culture, staining, and light microscopy.
Students should have prepared and observed wet mounts of Tetrahymena sp. and other microbes, noting distinguishing features.
Basic culture manipulation, including use of micropipettors, should be
familiar.
Student Activities:
·
Students will observe a live wet mount
preparation of Tetrahymena sp.
phagocytizing FITC-labeled prey (Saccharomyces
sp.) and complete Worksheet 1.
·
Students will analyze time study of Tetrahymena sp. phagocytizing
FITC-labeled prey by flow cytometry using Paint-A-Gate analysis of previously
acquired datafiles and complete Worksheet
2.
·
Students will work on Worksheet 3 in discussion groups during
lab propose possible questions about phagocytosis that could be addressed using
flow cytometry. Each group will formulate a question and describe a strategy of
investigation to share with the class. The class will decide which question(s)
to address and design an experiment
which will be conducted in the next laboratory.
Examples of experimental questions:
o
Does prey density determine the rate of
phagocytosis?
o
What is the optimum temperature for
phagocytosis? Salt concentration?
o
Do Tetrahymena
sp. exhibit prey preferences (size, cell type, etc.)?
o
Do Tetrahymena sp. eat more when they
are starved?
o
How many prey objects do Tetrahymena sp. ingest?
o
Do all Tetrahymena sp. ingest prey at the same rate?
o
What portion of the Tetrahymena sp. population phagocytizes?
Student Experiment:
·
Conduct the experiment on Tetrahymena sp. phagocytosis.
·
Analyze the experimental data using
Paint-A-Gate. The data will be compiled by the instructor and discussed in
class. A short laboratory report will be expected from each student.
Analysis of Tetrahymena and Phagocytosis Worksheet 1
Terms and Concepts:
·
Relative size
·
Relative complexity
·
Fluorescence
·
Autofluorescence
·
Fluorescence microscope
(image/qualitative)
·
Flow cytometry (relative
number/quantitative)
·
Forward scatter
·
Side scatter
·
FL-1
·
Datafile
·
Dot plot
Experimental question:
·
Does Tetrahymena pyriformis ingest yeast?
Experimental design:
·
Label Saccharomyces cereviseae with flourescein and see if Tetrahymena pyriformis ingests them.
Experiment:
·
Mix Tetrahymena
sp. with FITC-labeled yeast and determine if the prey had been phagocytized
using the fluorescence microscope.
Experimental observations and
speculations:
·
Observe the mixture of FITC-labeled Saccharomyces cereviseae and Tetrahymena
pyriformis. Be able to identify and describe the cells that are present.
o
Which cells, Tetrahymena sp. or yeast, are larger?
o
Which cells, Tetrahymena sp. or FITC-labeled yeast, generate higher forward
light scatter?
o
Which cells, Tetrahymena sp. or FITC-labeled yeast, are more granular?
o
Which cells, Tetrahymena sp. or FITC-labeled yeast, generate more side scatter?
o
Which cells, Tetrahymena sp. or FITC-labeled yeast, generate more fluorescence?
§
What population(s) of cells would you
see in each of the tubes below.
List each of the cells and draw
what you think they would look like
in terms of relative size,
complexity, and fluorescence:
|
Tetrahymena
sp. without FITC-labeled yeast |
FITC-labeled
yeast without Tetrahymena sp. |
|
Tetrahymena
sp. with FITC-labeled yeast at T=0 |
Tetrahymena
sp. with FITC-labeled yeast at T=’X’ |
Analysis of Tetrahymena and Phagocytosis Worksheet 2
Experimental question:
·
How long does it take Tetrahymena pyriformis to ingest food?
Experimental design:
·
Determine the time it takes for Tetrahymena sp. to ingest 50% maximum
food consumption.
‘Phagocytosis Time’ for
50% ingestion (or ‘PT50’).
Experimental controls:
·
Tetrahymena
sp. without FITC-labeled yeast.
·
FITC-labeled yeast without Tetrahymena sp.
·
Draw the expected results for each
control tube on the plots below (T=
Tetrahymena , Y= yeast ):
o
Control: Tetrahymena sp. without FITC-labeled yeast.
o
Control: FITC-labeled yeast without Tetrahymena sp.
Experimental samples:
·
Mix Tetrahymena
sp. with FITC-labeled yeast for the specified (‘X’) time, and determine if the
prey had been phagocytized using the flow cytometer.
·
Draw the expected results for each tube
on the plots below (T= Tetrahymena ,
Y= yeast ):
o
Tetrahymena
sp. with FITC-labeled yeast at T=0
o
Test sample: Tetrahymena sp. with FITC-labeled yeast at T=’X’
Experimental protocol:
·
Microbiology Service Center will
provide 12 X 75 Falcon 2054 tubes containing 25 ml
formalin, FITC-labeled yeast, and a well aerated 24-hour culture of Tetrahymena sp.
·
Students
should label 12 X 75 Falcon 2054 tubes (containing 25 ml
formalin) with your initials and the sampling times to be tested.
·
The instructor will add 100 ml
of Tetrahymena sp. without
FITC-labeled yeast, and 20 ml FITC-labeled yeast
without Tetrahymena sp. to each of two labeled control tubes containing 25
ml formalin.
·
The instructor will add (500 ml)
FITC-labeled yeast to a (3.0 ml, aerated) Tetrahymena
sp. culture, and obtain the T=0 control in a labelled 12 X 75 Falcon 2054 tube
containing 25 ml formalin.
·
Students
will transfer 100 ml of the Tetrahymena sp./ FITC-labeled yeast to each of their labeled tubes
(containing 25 ml formalin) at the specified time.
(Samples may be stored in the refrigerator until the next laboratory meeting
for flow cytometry.)
Sample acquisition on the flow
cytometer:
·
Students
should (recover their samples and) add 1.5 ml sheath fluid to each tube. (The instructor
will add sheath fluid to each of the three control tubes.)
·
Students
should take the tubes to the flow cytometer and (under instructor supervision)
acquire the data in list mode files for 10,000 events for forward light
scatter, side scatter and FITC/FL1 parameters.
·
Students
should save their data files for each time point on a ZIP disk that can be
transported to DH 550 for analysis using Paint-A-Gate PRO.
Data analysis using Paint-A-Gate PRO:
·
Students
should choose a sample time for analysis (facilitated by instructor).
·
Students
should open the FCS data files corresponding to their specified sample time in
Paint-A-Gate PRO:
o
Click on Bio 107.
o
Type in the password and wait.
o
Click on the icon, Items for ______________________________.
o
Click on the Program Paint-A-Gate icon.
o
If a box comes up asking whether to set
the monitor at 256 colors, click ok.
You should see
0.0 in a variety of colors at the top of the page.
o
Select File,
New PAG set. You will not see a
change on the screen.
o
Select Process, Load FCS file.
A box will appear on the screen.
o
Find the FCS data file corresponding to
your sample time: Click on the Desktop…….
(Ask the
instructor for the correct path and filename.)
·
Making Plots.
o
Select
and Open the FCS data file
corresponding to your sample time. Six plots will automatically appear
o
Close
all the plots except the SSC vs FSC plot by clicking on the box in the upper left
corner of each plot.
o
Drag
the plot to the upper left corner of the Desktop, select Display and Zoom In to
enlarge the plot. You should see something like this:
o
Make a new plot of SSC vs. FITC
fluorescence by selecting DISPLAY,
and New 2D plot. o
Change the X axis to FL-1H, and click OK. o
Drag
the new SSC vs FL1-H plot next to the SSC vs FSC plot. You should have two
plots: ·
Painting populations. o
Painting cell populations allows the
user to identify populations, and analyze them for various parameters. In this
case we want to know how many of the Tetrahymena cells contain FITC-labeled
yeast. Each of you should have 2 plots from a unique sample corresponding to
the sample time you selected. The plots should look pretty much like the ones
above. To find out which populations are the Tetrahymena cells, which are the
FITC-labeled yeast cells and which are Tetrahymena which have FITC-labeled
yeast cells in them, we can paint each population. The colored numbers below
the menu bar (all % = 0.0 above) will tell us the percent of the total cells
represented by the painted population. (All percents will be percent of TOTAL
events.) o
Click
on the left plot (to select it) and Select
Paint, then Red. Using the lasso
cursor enclose the population of cells of cells showing the highest SSC and FSC
(upper right of the plot on the left). The cells on the left plot will turn
red, and the cells in that population which appear on the plot on the right
evaluated for different parameters (SSC vs FITC-fluorescence/FL-H) will become
evident. What do the red and gray events represent? ·
Identify the events on the left plot: Red cells on the left
plot (SSC vs FSC):_______________________________________ ·
Identify the events on the right plot
by quadrant: Red events on the right
plot (SSC vs FL1-H): Upper
left quadrant (low FL1-H, high SSC):__________________________ Upper
right quadrant (high FL1-H, high SSC):________________________ What
percent of the total events are represented by the red events?_______ Gray events the right
plot (SSC vs FL1-H): Lower
left quadrant (low FL1-H, low SSC):__________________________ Lower
right quadrant (high FL1-H, low SSC):________________________ What
percent of the total events are represented by the gray events?_______ o
Click
on the left plot (to select it) and Select
Manipulate, then Exact Zap and Red. The red cell populations on both
plots should return to gray. o
Using the Paint and Zap functions
determine the percent of total events which are Tetrahymena cells which have
FITC-labeled yeast cells in them. §
Select
the right plot §
Paint
the upper left quadrant events red. §
Paint
the upper right quadrant events yellow. ·
What is the percent of total events
which are Tetrahymena
cells which have FITC-labeled yeast cells in them? ____________________ ·
Gating populations. o
Gating cell populations allows the user
to select specific populations, and analyze them for various parameters without
regard to the other events in the FCS data file. In this case we want to know
how many of the Tetrahymena cells contain FITC-labeled yeast without regard to
debris or the FITC-labeled yeast cells which are present but have not been
phagocytized by the Tetrahymena. The colored numbers below the menu bar (all %
= 0.0 above) will tell us the percent of the Tetrahymena cells (and only
Tetrahymena cells) represented by the painted populations. ·
Using the Gate, Paint and Zap functions determine the percent of
Tetrahymena cells which have FITC-labeled yeast cells in them. §
Manipulate
and Zap all events. All events
should be gray. §
Select
the left plot (SSC vs FSC). §
Paint
the upper right quadrant events (all Tetrahymena cells) red. §
Then Manipulate, Gate Events,
Red. Plots of SSC vs FSC and SSC vs
FL1-H will appear with only the Tetrahymena cells showing up (all gray).
§
Select
the right plot (SSC vs FL1-H), Paint,
and paint the low fluorescence,
Tetrahymena cells on the right plot red. §
Select
the right plot (SSC vs FL1-H), Paint,
and paint the high FL1-H events
yellow. ·
Identify the events on the left dot
plot (SSC vs FSC): Red
events:______________________________ Yellow
events:____________________________ §
Identify the events on the right dot
plot (SSC vs FL1-H): Red
events:______________________________ Yellow
events:___________________________ §
What is the percent of Tetrahymena
cells from
your sample (time) have FITC-labeled yeast cells in them? _________________ Compile data, discuss results and draw
conclusions: Analysis of Tetrahymena and Phagocytosis Worksheet 3 Experimental question: ·
Question on phagoctyosis in Tetrahymena pyriformis determined by lab
group: ______________________________________________________________________________? Preparation: ·
Do we have all the information we need
to conduct the experiment? ·
What additional information do we need? Experimental design: ·
Purpose of the experiment: ·
Controls: ·
Experimental groups: ·
Data expression/predictions: ·
Experimental protocol: ·
Materials from MSC: Experimental controls: ·
Draw the expected results for each
control tube on the plots below (T=
Tetrahymena , Y= yeast ): o
Control tube 1
contents:__________________________________________________________ o
Control tube 2
contents:__________________________________________________________ Experimental protocol: 2
________________________________________________________________________ 3
________________________________________________________________________ 4 ________________________________________________________________________ Compile data, discuss results and draw
conclusions: Answer to experimental question: o
___________________________________________________________________ Other important observations: o
___________________________________________________________________ o
___________________________________________________________________ o
___________________________________________________________________ o
___________________________________________________________________ Unanswered questions/next experiment? o
___________________________________________________________________ o
___________________________________________________________________
