20.109(F18):Evaluate cell loading experiment and treat cells for genomic instability experiment (Day3)

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20.109(F18): Laboratory Fundamentals of Biological Engineering

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Fall 2018 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Measuring genomic instability        2. Modulating metabolism        3. Engineering biomaterials              


Introduction

Today you will continue to examine the data collected for your cell loading experiment, and then use these results to probe the effects of a chemotherapeutic drug on genomic stability. DNA damage is defined as any change in the chemical structure of the molecule, including breaks in the backbone, missing basepairs, and altered basepairs. Damaged DNA does not mean mutated DNA! Though both instances relay that the DNA has been changed, a mutation is defined as a change in the sequence.

In lecture, you reviewed exogenous factors that lead to DNA damage (i.e. ultraviolet rays, smoking, etc.). Naturally occurring DNA damage results from native cell processes, such as metabolism. In this module, we will examine the effects of a chemotherapeutic drug on genomic stability using the CometChip assay.

Oxidative agent: hydrogen peroxide (H2O2)
Chemical structure of H2O2

Normal cell tissues have a basal level of DNA damage due to cell processes involved in cellular metabolism. For example, electrons can escape the electron transport chain and result in the formation of superoxide. Furthermore, defense mechanisms employed to protect the host from bacterial infection involved the release of reactive oxygen species. These reactive oxygen species are implicated in causing more than 20 types of DNA base lesions.



Protocols

Part 1: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on designing effective figures and captions.

Part 2: Image cell loading experiment

In the previous laboratory session you used the light microscope in the teaching laboratory to image your CometChip and determine the number of wells that were loaded and potentially how many cells were in each of the wells. Today you will use a fluorescent microscope in the Engelward Laboratory to confirm that enough cells were loaded to allow for the visualization of the DNA in the microcells using a florescent DNA dye. This will also allow you to see the microscope in the Engelward Laboratory, which will be used for all CometChip imaging in this module.

The teaching faculty will take one team at a time to the microscope in the Engelward Laboratory and image your CometChip. With the light microscope images, you determined the number of microwells that contained cells and potentially how many cells were present in the microwells. The images you examine today will give information on the number of wells that contain enough DNA to provide a fluorescent signal after staining. You can compare the number of microwells that contain cells from the light microscopy images to the number of wells that produce signal from the fluorescence microscopy images. Ideally, these numbers will be similar.


Part 3: Determine cell loading number for subsequent experiments

In a group discussion with the teaching faculty, you will assess the results of the class data from the CometChip loading experiments. The goal here is to determine which cell number to use when preparing your CometChip for the next experiments. Use the data you collected concerning the following: 1. number of wells loaded, 2. an estimate of number of cells per well, and 3. strength of DNA signal.

Be sure to include notes on the discussion and the values for cell loading number that you will use in your notebook!

Part 3: Treat cells for genomic stability experiment

Reagents list

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