Norfolk State University Assay Development Questions

1.Using your knowledge of the all biological (MTT, Cell Titer Glo, LDH) assays that you have learned in the class, explain how you will perform TWO different cell viability/toxicity assays using the single plate of cells that have been treated with the unknown compounds in the plate. 

1. First, identify the two cell viability/toxicity assays that you will apply (3 points)
2. Describe exactly what, biologically speaking, each assay is measuring (6 points)

2.For one of the assays that you selected, provide the following (6 points):

1. Control/s that will provide maximum signal 

a.Control/s that will provide minimum signal

b.Control/s that will provide background signal

M10

In the lecture you learned the different types of reporter assays/systems. Examples include CAT, beta galactosidase, SEAP, luciferase, GFP, FlAsH/ReAsh and beta lactamase. (20 points)

1.If you wanted to study interaction between protein A and protein B in cell-based assay, which of the reporter system would you use and why, how would you set up the assay and types of controls you would require (10 points)

2.If you wanted to test if your compound is activating NF-kB signaling, which reporter system would you use and why, how would you set up the assay and types of controls you would require (10 points)

M11

Apoptosis is a genetically programmed process of eliminating unwanted or abnormal cells in the body. Dysregulation of apoptosis has implications in the development of diseases such as cancer, AIDS, autoimmune, and neurodegenerative/neurodevelopmental diseases. This diversity of diseases associated with apoptosis stresses the need to identify proteins that are involved in  programmed cell death. Apoptotic cells exhibit certain morphological and biochemical features that can serve as markers for detection. Activation of specific caspase cascade  is considered  a direct and specific marker for identifying apoptosis.  Recent studies have shown that apart from death receptor and mitochondrial apoptosis pathways, other cellular  organelles also  initiate specific apoptosis pathways. Although these pathways originate in an organelle-specific manner, they all converge on the central executioner, which is the activation of the caspase cascade.   

So knowing the importance of dysregulation of apoptosis in diseases and having learnt in the lecture the different hallmarks and assays of cells undergoing apoptosis, answer the following questions: 

1.Describe two cellular apoptosis assays that will utilize High content imaging to monitor apoptosis (ex. Caspase activation and flip-flopping of PS). The two assays could be multiplexed or you can set up as two independent assays (10 points) 

2.Describe the different types of controls you will need to set up  (6 points)

3.How will you validate the assay (4 points)