Difference between revisions of "20.109(F18):Prepare and treat cells for genomic instability experiment (Day3)"

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.

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: Determine cell loading number for genomic instability experiment

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: Harvest cells for genomic instability experiment

Part 4: Load cells into CometChip

1. You will load CometChips prepared by the teaching faculty, but first need to calculate the volume of cell suspension to load using the following information:
   • For the cell number, you will use the value selected during the group discussion today.
   • The cell suspensions (both wild +DNA-PK and -DNA-PK) prepared by the teaching faculty is at a density of 500,000 cells / mL.
2. Retrieve your CometChips from the front laboratory bench.
3. Obtain one glass plate, one 96-well bottomless plate, and four 1.5" binder clips from the front bench per CometChip.
   • Each partner will complete the following steps for one of the CometChips such that both partners are completing the steps in parallel.
4. Remove your CometChips from the 1x PBS and place it, gelbond side down, on the glass plate. Save the dish your CometChip was stored in for storage later.
5. Press the 96-well bottomless plate onto the CometChip so that the wells line up with your labeling as shown in the diagram on the right.
   

   

   Plate map for biochemical testing experiment.
   • Be sure to press the top of the 96-well bottomless plate onto the CometChip. If you are unsure which side is the top, please ask the teaching faculty.
   • Do not move the 96-well bottomless plate while it is on the CometChip as you will damage the agarose and the microwells.
6. Use the binder clips to secure the 96-well bottomless plate to the glass plate, thus creating a 'sandwich' with your CometChip in the center.
   • Fasten the binder clips to the very edge of 96-well bottomless plate.
7. Add the appropriate volume of your wild-type cell suspension (calculated in Step #1) to the wells in Rows A, B, and C.
8. Add the appropriate volume of your mutant cell suspension (calculated in Step #1) to the wells in Rows D, E, and F.
9. Incubate your CometChip in the 37 °C incubator in the main laboratory for 15 min.
10. After the incubation, complete a wash step to remove excess cells that are not within the microwells of your CometChip.
    • Carefully remove the binder clips and the 96-well bottomless plate.
    • With the CometChip on the glass plate, 'waterfall' ~5 mL of 1x PBS over the wells, which are now imprinted onto the agarose.
      • Hold the glass plate with the CometChip such that the letter labels are at the top to ensure you do not wash cross-contaminant the cells in the wells.
      • To waterfall the 1x PBS, hold the glass plate at a 45° angle over a blue plastic dish, found at front bench.
      • Pipet up ~5 mL of 1x PBS.
      • Press the pipet tip onto the glass plate above your CometChip.
      • As you expel the 1x PBS, quickly move the pipet tip from left-to-right.
      • The 1x PBS should pass over the top of the CometChip and fall into the dish.
    • Use a P200 tip attached to the pasteur pipet to aspirate the excess liquid from your CometChip wells.
      • Lightly touch the tip to the bottom of each imprinted well on the CometChip and immediately lift the tip from the agarose.
11. Retrieve one tube of molten 1% low melting point (LMP) agarose from the 42 °C waterbath.
    • You will need to work quickly from this point as the LMP agarose will solidify as it cools.
12. Using the P1000, pipet up 1000 μL of molten agarose from the tube.
13. Hold the pipet tip over the top left well of your CometChip and as you expel the agarose move the pipet tip from left to right. Ensure that each row of your CometChip gets covered.
    • The goal is to lightly cover the wells that contain cells, which will 'trap' the cells into the microwells.
    • If the LMP agar 'fell' off the CometChip in any areas during this process, it is important to 'fill in' those portions of the CometChip. Please alert the teaching faculty if you experience any difficulties!
14. Leave your CometChip undisturbed on the benchtop for 3 min then carefully move it to the 4 °C cooler for 5 min to ensure the LMP agarose solidifies.

Part 5: Treat cells for genomic instability experiment

Reagents list

Navigation links

Next day: Load cells for genomic instability experiment and induce oxidative stress