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Flow Cytometry at San Jose State University

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Analysis of Tetrahymena and Phagocytosis Using

Fluorescence Microscopy and Flow Cytometry

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:

 

  1. ________________________________________________________________________

 

 

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       ___________________________________________________________________

 

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