DIVA Tutorial

DIVA Tutorial
DIVA Tutorial
Log in using your PI as username. No password is required.
When you open DIVA, you will see the following five windows:
1. Browser
From this window you can:
a. Open a new or existing experiment.
b. Duplicate an experiment without data.
c. Store a Template.
d. Export data.
2. Cytometer
a. Add or delete fluorophores (Parameter Tab).
b. Set voltages (Parameter Tab).
c. Choose fluorophore parameters (Log/Linear Scale, Area, Width, Height) - (Parameter
Tab).
d. View and/or edit compensation (Compensation Tab).
3. Acquisition Dashboard
a. Acquire and record data.
b. Set up number of events to record.
c. Tell machine what gate to count from when recording.
d. Monitor threshold and number of events acquired.
4. Inspector
a. Provides information and options to make changes on whatever it is you are clicked on.
b. If clicked on a worksheet/graph:
i. Label graphs.
ii. Assign a population to be viewed in the graph.
iii. Change axis to bi-exponential.
c. If clicked on a tube:
i. Label tubes.
ii. Number of events collected for this tube.
d. If clicked on an experiment:
i. View fluorophores and voltages used.
ii. View compensation.
iii. Print out parameters.
5. Worksheet
a. Setup analysis/acquisition template.
b. Add plots and graphs.
c. Add Statistics.
Experiment Set-up
The Browser Icons/Commands Overview
a. Experiment
i. The icon is a notebook, when the experiment is closed, the book is closed.
When the experiment is open, the book is open.
ii. The experiment must be open in order to add/delete tubes, make a worksheet,
add fluorophores, or view data.
iii. Open an existing experiment in the browser by double clicking on the icon or by
right clicking on it and choose the Open Experiment option.
b. Worksheets
(Normal vs. Global)
i. Every new experiment will automatically provide you with one global
worksheet.
ii. To add additional worksheets, right click the Global Worksheet icon and choose
New Global Worksheet.
iii. Global worksheet (default worksheet used for samples): All tubes will have the
same gating strategy, no changes from tube to tube. If you move the gate it will
be applied to all samples.
iv. Normal Worksheet (used for Compensation Controls): Specific to each tube.
Every tube will have a separate worksheet that allows for separate gating
strategies per sample tube.
v. Toggle between Normal and Global worksheets by clicking on the far left icon
located on the Worksheet toolbar.
c. Specimen
i. This is where you organize your tubes to be analyzed on the Flow Cytometer.
d. Tubes
i. These are your samples that will be run on the flow cytometer.
ii. Tubes are added underneath the Specimen Icon in an experiment.
iii. Add tubes by clicking on the Tube icon, right clicking on an existing tube and
choose Duplicate Without Data, or by clicking Next Tube in the Acquisition
Dashboard.
e. Cytometer Settings
i. Clicking on the cytometer settings will allow you to view your parameters
(voltages, fluorochromes, compensation) in the inspector window.
ii. View whether or not you have a compensation linked to the experiment:
iii. Add analysis templates and/or application settings.
f. Duplicate without Data
i. This function allows you to duplicate an entire experiment, specimen or tube.
ii. All of the labels, parameters, set-up will be carried over when using this tool.
iii. Right click on Experiment, Specimen, or Tube to locate this feature.
Creating a New Experiment
1. Go to Experiment on the Tool Bar Menu and choose New Experiment
2. Select Blank Experiment with Sample Tube, Click OK
3. A new Experiment icon will appear in the browser:
a. Right Click on the experiment to rename.
4. Click on the “+” icon to the left of the Experiment to expand.
5. You’ll see the following sections are included in your experiment: Cytometer Settings, Global
Worksheets, and Specimen.
a. You can expand each section by clicking on the “+” icon to the left.
Specimens and Tubes
6. Add your tubes/specimens and label
a. Right click on the Specimen and choose New Tube or highlight Specimen and click on the
tube icon.
b. Label tubes and specimens.
c. Take a moment to look at the Cytometer and Acquisition Dashboard Windows – Notice
that you only have one tab available in the Cytometer window and the Acquisition
functions are grayed out.
d. To the left of each tube is an arrow icon, click on the arrow to activate the tube – the
arrow will turn green (alternatively you can double click the Tube icon).
e. Notice that you now have more options available in the Cytometer and Acquisition
windows now that a Tube is “active”.
f. THE ARROW TO THE LEFT OF A TUBE MUST BE GREEN IN ORDER TO ACQUIRE OR SET
UP PARAMETERS. IF THE TUBE IS NOT ACTIVE THEN YOU WILL NOT BE ABLE TO SETUP
YOUR EXPERIMENT OR COLLECT EVENTS.
Fluorophores
7. Add Fluorophores
a. Click on a Tube to make it “active”.
b. Click on the Parameters tab in the Cytometer Window.
c. Delete all of the fluorophores, but leave the FSC and SSC (First two listed).
i. Click on the “dot” to the left of a fluorophore to select. Hold down the Shift key
to select multiple fluorophores and click the Delete button.
d. Click on the Add button.
e. Click within the fluorophore box to get a drop-down menu of available options.
f. Choose the fluorophore you need.
g. Repeat above steps to add additional fluorophores.
h. Verify that the FSC and SSC have the Log function unchecked and A, H and W checked.
i.
j.
Make sure that each fluorophore has the “A” function checked.
A = area is the sum of all pulse heights, H = maximum height value for the pulse, and W
= width of the pulse (calculated by the instrument’s electronics).
Compensation Controls
8. Create Compensation Controls (if applicable).
a. Click on Experiment in the Tool Bar Menu.
b. Select Compensation Setup and choose Create Compensation Controls
c. A dialogue box will appear:
i. Verify that all your fluorophores are listed, “include separate unstained control
tube” is checked, and the label for each fluorophore is “Generic”.
ii. If you have labels associated with a tube, simply delete the ones with a label
and leave the generic ones.
iii. Click OK.
d. A “Compensation Controls” Specimen will be in your experiment
i. Expand the Specimen and you will see that Tubes and Normal Worksheets have
been created for each Fluorophore in your experiment.
Worksheet
9. Create Worksheet
a. When acquiring on the LSR, it is best to keep the worksheet simple. You can analyze and
make more graphs/plots during analysis using one of the Flow Core workstations
(FlowJo and DIVA).
b. Once data is acquired you can add and delete plots/gates as much as you want using the
FSC files. VOLTAGES CANNOT BE CHANGED on FSC files. Voltages need to be
determined during acquisition.
c. The Tool Bar on the Worksheet window contains icons for graphs and gates. Hold the
mouse over the icon in the tool bar to view what type of gate or graph you are choosing.
i. Graphs
1.
2.
3.
ii. Gates
1.
2.
3.
4.
Dot Plot
Contour Plot
Histogram
Polygon
Rectangle
Interval
Quadrant
Plots
d. To add a plot to the workspace, click on the plot type that you want and then click
within the worksheet space.
e. Once you have chosen the appropriate plots, click on the axis to choose the
fluorochrome of interest that you want displayed on the plot.
f.
Right click on a graph and choose Show Population Hierarchy
i. This allows you to track what is being shown in each plot.
ii. You will also be able to get the % of each population.
Gates
g. Click on the gate type (polygon, rectangle, interval, or quad) and then click back in the
plot of interest. The gate will appear. You will also see that it has been added to your
Population Hierarchy with a color (red) and name (P1) assigned.
h. To add a gate based on your P1 data:
i. Click on the “P1” gate in the Population Hierarchy box.
ii. Click on a gate icon.
iii. Click on the appropriate plot. You’ll see the gate appear with a P2 label, you’ll
also see that it has been added to the population hierarchy with a color (green)
and label (P2) assigned.
iv. Continue with the above process to add the appropriate gates to your
workspace.
Plot Populations and Options
i.
Next you want to tell the software what populations to show in each plot
i. Right click on a plot
ii. Select “Show Populations”
iii. Pick the population you want the plot to depict (This particular plot is showing
“All Events”)
j. Rename Populations
i. Double click on a population in the Population Hierarchy box
ii. Type name
k. Assign/Change color of a population
i. Right click on the color box located to the left of the population in the Hierarchy
box
ii. A drop down menu of colors will appear, click on the color you want
l. Label your plots so that you know what population each one is showing
i. Select all the plots by holding down the shift key and clicking on all the plots
ii. Go to the Inspector Window and choose the Title tab
iii. Select the Tube and Populations boxes
m. Add statistics
i. Right click on a plot
ii. Choose Create Statistics View
iii. A statistics box will appear on the workspace
iv. Right click on the statistics box and choose “Edit Statistics”
v. Pick the parameters you are interested in: Mean, Median, Etc.
n. Change an axis to Bi-exponential
i. Click on the plot
ii. Go to the Inspector Window
iii. Select on the Plot tab
iv. Under the Bi-exponential Display section, check mark the appropriate axis
Acquisition
10. Setup Acquisition Dashboard
a. Go to Acquisition Dashboard Window
b. Set the number of Events to Record
c. Choose the Stopping Gate
i. This is the gate the machine will use to count the number of events
d. Acquire Data Button: Acquires data points that you can see in the worksheet
e. Record Data Button: Collects data for the FCS files
f. Storage Gate: Leave this set on All Events
Data Management
11. Export Data
a. Batch Analysis: Provides a PDF. You cannot move gates or make any changes to a Batch
Analysis file.
i. Right Click on either the Experiment icon or Specimen icon to batch the report
ii. Clicking on the Experiment icon will give you all of the analysis worksheets for
every tube in the experiment.
iii. Clicking on the Specimen icon will batch only the tubes associated with the
specimen
iv. Select Batch Analysis (In this case we are batching the entire experiment)
v. A dialogue box will appear
vi. Check the Output to Printer and Save as PDF options
vii. Click on Browse and navigate to your Jump Drive
viii. Click Start
ix. Click OK to the pop- up windows
x. Print
b. Export FCS Files: Exports FCS files that can be imported into DIVA, FlowJo or other flow
analysis software. You can change gates and plots, add statistics and create PDF files.
i. Right click on the Experiment.
ii. Choose Export.
iii. Click on Experiment or FCS File (v 3.0)
a. Experiment: You will save all worksheets associated with the
experiment and the parameters.
b. FCS Files: Saves data files only. No worksheets will be carried
over when you open the files.
iv. Navigate to your Jump Drive and Save.
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