1st Assignment (30 Points)
Covers Material Presented in Units 1-3
Section I. (20 Points Total)
The following Topics 1-3 (of 4) cover the cell isolation protocols from Units 1 & 2: Assigned Appendix readings; Isolation of human monocytes by double gradient centrifugation; the Rosette Sep ™ isolation method; and protocols using MACs technology. Your answers should explain the main techniques in your own words. For example: In this protocol the T cells were isolated by incubating them with a CD3-specific antibody that was conjugated with biotin. Following incubation, the excess antibody was removed by washing with buffer and centrifugation. Common protocol details, such as speeds and duration of centrifugation, are not required (e.g., Spin the cells for 30 minutes at 3,000 RPM).
Topic 1. (10 points) Monocyte Isolation
- (2 points) What is a buffy coat? How is it acquired?
- (2 pts) In the monocyte isolation assay it is mentioned that heparinized peripheral blood can be substituted for buffy coats. What is heparin? Explain why the peripheral blood is heparinized.
- (4 points) In the monocyte isolation assay, they authors state that the “resulting resting macrophages…are still capable of responding in either an M1 or M2-like fashion.” Do a little research and explain: 1) what do the authors mean by “resting macrophages” and 2) what are the functional differences between M1 and M2 macrophages.
- (2 points) The monocyte isolation used two types of isolation procedures to enrich the final macrophage population (i.e., remove other cell types such as lymphocytes). The first is density gradient. What is the second?
Topic 2. (5points) RosetteSep isolation method (Be sure to watch the video)
In the RosetteSep isolation method, RBCs were not removed before isolation of the cell type of interest.
- (2 pts) Explain how the RBCs played an important role in the isolation protocol.
- (3 pts) Explain the biological mechanisms involved: find out what is contained in a particular RosetteSep solution and explain the function of the different components within the solution.
Topic 3. (5 points) MACS Technology:
- (3 pts) Explain the difference between negative and positive selection isolation methods according to MACS protocols.
- (2 pts) In the MACs eosinophil isolation protocol why were the pelleted RBCs lysed after Ficoll-Paque™ gradient cell separation rather than simply discarded after the first centrifugation?
Section III. (10 Points) Antibodies for Research and Diagnostic Tools
The following questions are based on Magic Bullets and Monoclonals, FASEB Break Throughs in Bioscience; Appendix I, A-5—A12;
Topic 1. (3 points)
Contrast and compare the work of Koch and his “postulates” and the work of Behring that identified the existence of antibodies. How was Behring’s work similar and dissimilar to the work of Robert Koch that resulted in Koch’s development of his “postulates”?
Topic 2: (2 point)
Why was it difficult to isolate the antibodies specific for a disease from the blood of an inoculated animal?
Topic 3. (5 points)
Today, monoclonal antibodies are being successfully used to treat certain cancers (e.g., Rituximab). However, Nadler’s and Stashenko’s first attempt to treat B cell lymphoma with a monoclonal antibody was a failure.
- What was the target of this first antibody? Explain why this first antibody failed. (3 points)
- Monoclonal antibody treatments, such as rituximab (Rituxan), induce the destruction of cancer cells. Describe two natural immune mechanisms that can account for rituximab’s success as a therapy. (2 points)