MLS460 Immunology_Lab Assignment 1

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CUNY Hunter College *

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460

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Biology

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Feb 20, 2024

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MLS460 Immunology Dr. Abigail Morales Instructions for Lab Assignment 1 DUE: Monday, March 6, 2023 by 11:59pm via Turnitin on Blackboard Part 1: Griess Assay (30 pts total) In the Griess Assay, each group generated a data set for a specific set of unknown samples (as well as a standard curve using NaNO 2 ): Group 1: Untreated Heat-killed Listeria monocytogenes + IFN- β Untreated + iNOS inhibitor Heat-killed Listeria monocytogenes + IFN- β + iNOS inhibitor Group 2: WT Untreated WT Listeria monocytogenes Myd88 -/- Untreated Myd88 -/- Listeria monocytogenes Group 3: WT Untreated WT Listeria monocytogenes Ifnar1 -/- Untreated Ifnar1 -/- Listeria monocytogenes Group 4: Untreated IFN- β Heat-killed Salmonella typhimurium Heat-killed Salmonella typhimurium + IFN- β Provided on Blackboard in Excel files: • The data sets generated by all four lab sections. The values given in the table are the OD548 for each sample (standard curve and unknowns). • A Representative Data Set generated by your instructor (OD548 values for a standard curve and for each unknown in the above four groups). Tasks: 1. Graph the standard curve for YOUR GROUP’S DATA SET. Use your graph to determine the nitrite concentration of each unknown sample in that data set (2.5 pts). 2. Generate a data table for your group’s data set that indicates the concentration of nitrite in each of the unknown samples (2.5 pts).
3. Graph the standard curve for the Representative Data Set . Use your graph to determine the nitrite concentration of each unknown sample in the data set (i.e. the unknowns for all four groups) (2.5 pts). 4. Generate a data table for the Representative Data Set that indicates the concentration of nitrite in each of the unknown samples (2.5 pts). Questions: 1. (1 pt) Is your group’s standard curve perfectly linear? Explain (and please be specific). 2. (3 pts) Do your group’s results agree with the Representative Data Set? Briefly discuss. 3. (2 pts) Based on the results you obtained from analyzing the Representative Data Set, is the iNOS inhibitor effective at blocking the effects of the iNOS enzyme? Why or why not? 4. (2 pts) What is MyD88? Do you expect signaling downstream of Toll-like receptors (TLRs) in Myd88 -/- macrophages? Do you expect nitric oxide production by these cells? Briefly explain. 5. (2 pts) Do Ifnar1 -/- macrophages infected with Listeria monocytogenes produce type I interferon? Do they up-regulate iNOS expression? Explain your reasoning. 6. (3 pts) Does treatment with heat-killed Salmonella typhimurium alone result in nitric oxide production? Does this result agree with what you expected? Why or why not? 7. (3 pts) Nitric oxide production is a reliable indicator of classical macrophage activation. Name 3 other indicators of macrophage activation. 8. (3 pts) With your response to #7 in mind, design an experiment by which you could assess whether or not a macrophage is activated. Note : it must be different than the experiment we performed in class. Please include a minimum of TWO references (aside from your text or the lab handout) for this section (1 pt). Part 2: IL-6 ELISA (20 pts total) In the IL-6 ELISA experiment, each group generated a data set for a specific set of unknown samples (as well as a standard curve using mouse recombinant IL-6): Group 1: WT Untreated WT Listeria monocytogenes Il-6 -/- Untreated Il-6 -/- Listeria monocytogenes Group 2: WT Untreated WT Listeria monocytogenes Ifnar1 -/- Untreated Ifnar1 -/- Listeria monocytogenes
Group 3: Untreated LPS Untreated + anti-IL-6R LPS + anti-IL-6R Group 4: Untreated Poly I:C Heat-killed Staphylococcus aureus Provided on Blackboard in an Excel file: • A Representative Data Set generated over the course of the three lab sections. Shown are OD450 values for a standard curve and for each unknown in the above four groups. Tasks: 1. Graph the standard curve for the Representative Data Set . Use your graph to determine the IL-6 concentration (pg/mL) of each unknown sample in the data set (i.e. the concentrations of the unknowns for all four groups). Your curve may not be perfectly linear; that is OK (2.5 pts). 2. Generate a data table for the Representative Data Set that indicates the concentration of IL-6 in each of the unknown samples (again, for all four groups) (2.5 pts). Questions: 1. Describe a “capture” or “sandwich” ELISA (2 pts). 2. What is the purpose of the blocking step in an ELISA (1 pt)? 3. Why is biotin-avidin chemistry so widely used in commercially available ELISA kits (1 pt)? 4. IL-6 is a key pro-inflammatory cytokine. What are some of its roles in the immune response? (1 pt) 5. Did IL-6 -/- macrophages produce IL-6 after infection with Listeria monocytogenes ? Was this result what you expected? Why or why not? (3 pts) 6. Is type I interferon signaling required for IL-6 production during Listeria infection? Please comment specifically on the data when answering the question. (1 pt) 7. Did macrophages treated with both LPS and anti-IL-6R produce IL-6? Was the result what you were expecting? Why or why not? (3 pts) 8. Which TLR stimulus produced more IL-6, poly I:C or heat-killed S. aureus ? What can you conclude from this result? (2 pts) Please include a minimum of TWO references (aside from your text or the lab handout) for this section. (1 pt)
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