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SIV - Experiment Analysis Standard Operating Procedure

Data courtesy of Dr. Joern Schmitz, Beth Israel Deaconess Medical Center, Inc.

Purpose & Hypothesis: Detection of T-cell responses following administration of an AIDS vaccine to rhesus monkeys.

2.2.3.1 SIV Use Case Description
3.2.3 SIV Results
4.2.3 SIV Discussion

Monkeys were immunized with the peptide multiple times over the course of the study. The first run was PRE immunization. Every run has files for 3 primates, each stimulated in vitro with peptide, positive control, or left unstimulated, resulting in 9 test samples per run. variables

 

 

You will be looking for any Cytokine response (IFN, IL2, TNF) of the T cell populations (CD4 or CD8) in the samples, to determine the effectiveness of the peptide treatment as compared to the unstimulated sample and the positive treated sample.  Each Timepoint has its own workspace.  The analysis steps will have to be repeated for each of the six HIV sample run workspaces.

 

 

  1. Open FlowJo version 7.5 or later, not v8 or v9. Earlier versions will not be able to perform this analysis. Open the FlowJo a SIV workspace file. (File>open workspace). Do not remove any tables, layouts, or stat nodes. Do not change transformation settings or compensation matrix settings.
  2. Click on the group “TUBE NAME= Monkey3” in the upper Groups and Analyses window.
  3. Click the drop-down arrow next to the “UNSTIMULATED_run1_monkey3.fcs” file in the lower part of the workspace window.
  4. Double click on “UNSTIMULATED_run1_monkey3.fcs” “Lymphocytes,” “Singlets,” “CD3,” “CD4+,” and “CD8+” to get pop-up windows of each of the graphs. Change the Y-axis of the CD8+ graph window to “Histogram” and arrange as shown below.
  5. In all of the graphs except CD4+ and CD8+, there will be one or two gates encompassing populations of cells (dense populations are indicated by red, yellow and green dots). Some of the of the gates are not around the populations they should be, and must be adjusted to encompass the appropriate areas. This task must be done in order—first check the gate on the unstimulated sample, then the lymphocytes, singlets, CD3, and CD4+ and CD8+. The order is represented by the numbers in the figure above.
  6. With the histograms CD4+ and CD8+, change the X-axes to each of the following parameters “Comp-APC-A:: IL2,” “Comp-FITC-A:: TNF,” and “Comp-PE-CY7-A:: IFN”—this can be done using the drop-down menu shown in the figure below. Check that the range gate (as shown by tbe horizontal line) extends from the right-hand side of the window to the right base of the peak for each view.
  7. Leave these windows open and return to the Workspace window. Hold “shift” and select the entire gate tree under the sample you edited, from “Lymphocytes” through “Count.” Drag these gates on to the “TUBE NAME= Monkey3” group to apply the gates you modified to the other samples in the same primate. If you were successful, all the gates should change to the color of the primate group (yellow, in this case).
  8. Go back to your gate windows. In the upper right corner of the UNSTIMULATED graph window, click one of the next or previous arrows while holding the SHIFT key.  This will scroll through the other windows and allow you to view the same gates in the samples “peptide_run1_monkey3” and “positive_run1_monkey3.” Adjust the gates as you did in steps 5 and 6.
  9. Repeat these same steps for the other two monkeys, “Monkey2” and “Monkey1.”
  10. Save your workspace on the provided flash drive with this example nomenclature: SIVrun1_Name.wsp.  When the FlowDx DB is funtional,  clicking “Save” will automatically save your workspace into the repository with all the correct information.
  11. Repeat these steps for each of the six SIV sample run workspaces.