From Course Wiki
Jump to: navigation, search
20.109(F22): Laboratory Fundamentals of Biological Engineering

Fa22 banner image v3.png

Fall 2022 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Genomic instability        M2: Drug discovery        M3: Project design       


For the next experiment in this module you will use the CometChip assay developed in the Engelward Laboratory. The CometChip assay is used to assess various types of DNA lesions, including base excisions, abasic sites, strand breaks, and crosslinks.

Microscopy images showing results from CometChip assay. A. In the CometChip assay DNA damage is measured by determining the amount of DNA in the head versus the tail of the comet. B. Cells not treated with DNA damaging agent (left) have visibly less DNA damage that cells treated with DNA damaging agent (right) as evidenced by the amount of DNA in the tail.
To measure DNA damage, the CometChip assay relies on gel electrophoresis. Electrophoresis is a technique used to separate molecules by size using an applied electrical field and a sieving matrix. DNA, RNA and proteins are often studied with this technique; agarose and acrylamide gels are the two most common sieves. The molecules to be separated enter the matrix through a well at one end and are pulled through the matrix when a current is applied. Because DNA and RNA are negatively charged molecules due to their phosphate backbone, they naturally travel toward the positive charge at the far end of the gel. Larger molecules are entwined in the matrix and are stalled; smaller molecules wind through the matrix more easily and travel further from the well. Over time fragments of similar length accumulate into 'bands' in the gel. The CometChip assay is based upon the principle that damaged DNA travels more readily compared to undamaged DNA.

In the CometChip assay, cells are loaded into microwells that are 'stamped' onto an agarose gel sieving matrix. Specific treatments can then be applied to the cells within these microwells that induce DNA damage. The cells are then lysed to release the DNA into the microwell. Following cell lysis, the CometChip is incubated in an alkaline buffer that unwinds the DNA. This step allows for all types of DNA damage to be detected. Lastly, gel electrophoresis is used to separate the DNA fragments. DNA fragments migrate away from the microwell and generate a comet tail as shown in the image to the right (panel A). The distance that the DNA migrates (i.e. the length of the comet tail) is proportional to the amount of damage. For example, with cells not treated with a DNA damaging agent, there are no evident comet tails to the right of the microwell or 'head' (panel B, image on the left); however, in cells treated with a DNA damaging agent there are comet tails apparent (panel B, image on the right). Comet tail lengths can be compared across experimental treatments to determine the deleterious effects of chemicals and toxins on DNA stability. In addition, the CometChip assay can be used to study the rate and efficiency of repair in response to specific treatments.


Part 1: Participate in Communication Lab workshop

Our communication instructor, Dr. Chiara Ricci-Tam, will join us today for a discussion on writing figure titles and captions.

Part 2: Perform CometChip experiment

As with the γH2AX experiment, the MEF cells with be treated with H2O2 +/- As exposure. The treatments will be applied in two parts. First, cells will be incubated in As for 2 hrs to mimic exposure to the toxic metal. Second, the cells will be incubated in H2O2 for 30 min to induce DNA damage. Following treatment with H2O2 the cells will be visualized to assess DNA repair.

To ensure the steps included below are clear, please watch the video tutorial (linked here). The steps are detailed below so you can follow along! Please note that the video shows the general steps for completing an experiment using the CometChip assay. The specific procedure used to generate the data you will analyze are written below. Furthermore, the video shows two version of the CometChip assay: the alkaline and the neutral. In this module, the alkaline CometChip assay was used.

Expose cells to As
Due to the timing of the As treatment, the Instructors completed the below steps using MEF cells that were seeded in T25 flasks 48 hr prior to the laboratory session. Using the As treated cells you will complete the steps starting at #3.

  1. Aspirate spent media from flasks.
  2. Add 0 μM, 10 μM, or 40 μM As-containing media to the appropriate flask and incubate at 37 °C for 2 hours.
  3. Retrieve flasks from the incubator and remove As-containing media.
    • Remember to collect all As waste in the appropriate waste stream!
  4. Wash the cells by adding 3 mL PBS using a 5 mL pipet.
  5. Slightly tip the flasks back and forth to rinse the cells then aspirate the PBS with a Pasteur pipet.
  6. To dislodge the cells from the flasks, add 1 mL of trypsin to the flask with a 2 mL pipet.
  7. Tip the flasks in each direction to distribute the trypsin evenly then incubate the cells at 37 °C for 2 minutes.
  8. Retrieve your flasks from the incubator and firmly tap the bottom to dislodge the cells.
    • Check your cells using the microscope to ensure they are dislodged. They should appear round and move freely.
    • If your cells are not detached from the flask, incubate at 37 °C for an additional minute.
  9. When your cells are dislodged, move your flasks back into the tissue culture hood and add 3 mL of media to the cells then pipet the liquid up and down (“triturate”) to break up cells that are clumped together and suspend them in the liquid.
  10. Transfer the suspended cells from each flask into labeled 15 mL conical tubes.
    • Each suspension should be transferred into a separate conical tube!
  11. Aliquot 90 μL of each cell suspension from the 15 mL conical tube into a labeled eppendorf tubes.
  12. Add 10 μL of trypan blue to the aliquots and count the total number of cells in each of your flasks.
    • The total number of cells = the calculated value for cells per mL * the total volume of the cell suspension.
  13. To remove the media containing serum prior to H2O2 treatment, pellet the cell suspensions at 500 g for 3 min then carefully aspirate the media leaving just the cell pellet in the bottom of the conical tube.
    • Be very careful at this step as it is easy to aspirate the cells if the Pasteur pipet is too close to the pellet.
  14. Calculate and the appropriate volume of serum-free media to achieve a cell suspension density of 500,000 cells/mL for each of your As-treated cell preparations.

Load cells into CometChip

  1. Remove your CometChips from the 1x PBS and place, gelbond side down, on separate glass plates.
    Binder clip placement for CometChip sandwich.
    • You will load two CometChips in this step! The cells in one of the CometChips will be treated with H2O2 and the cells in the the other will be used as a no treatment control.
  2. Press a 96-well bottomless plate upside-down onto each CometChips.
    • Be sure to press the top of the 96-well bottomless plate onto the CometChip.
    • Do not move the 96-well bottomless plate while it is on the CometChip as you will damage the agarose and the microwells.
  3. 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 as shown in the image to the right.
  4. Calculate the volume of cell suspension that contains 50,000 cells given the density of the cell suspension is 500,000 cells / mL.
  5. Add the appropriate volume of each cell suspension calculated in the previous step into each of the macrowells according to the plate map below (load cells into the grey color-coded macrowells).
    Fa22 cometchip plate map.png
  6. Cover the top of your CometChip with plastic wrap then incubate in the 37 °C incubator in the main laboratory for 15 min.
  7. After the incubation, complete a wash step to remove excess cells that are not within the microwells of your CometChip. Read all the bullets below before proceeding!
    • 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.
      • Hold the glass plate at a 45° angle over the dish that you used to store your CometChip.
      • Pipet up ~5 mL of 1x PBS.
      • Press the pipet tip onto the glass plate above your CometChip.
      • As you expel the 1x PBS, 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.
  8. Use the molten 1% low melting point (LMP) agarose is located in the 42 °C waterbath on the front laboratory bench to 'trap' the cells in your CometChip.
    • You will need to work quickly from this point as the LMP agarose will solidify as it cools.
  9. Using the P1000, pipet up 1000 μL of molten agarose from the bottle.
  10. 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.
  11. 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.
  12. Cells loaded into your CometChip will cluster into microwells (see image below).
Microscopy image of loaded CometChip. (A) Bottom of macrowell showing evenly spaced microwells. (B) Close-up of empty microwell. (C) Close-up of microwell loaded with cells.

In your laboratory notebook, complete the following:

  • Include the calculations completed to determine the volume of cell suspension added to each macrowell for the CometChip assay.
    • The cell suspension is 500,000 cells / mL and 50,000 cells were added into each well.
  • Given the number of cells that were loaded into each macrowell, estimate the number of cells per microwell.
    • Consider the likelihood that every cell you add to the well will fall into a microwell.
    • Perhaps calculate the surface area of the bottom of a well (of diameter 6.35 mm) and compare this to the size of the cells (~10 μm).
    • Also, remember that within each macrowell there are ~ 300 microwells, which are ~40 μm in diameter and 40 μm in depth.

Treat cells with H2O2

  1. While the LMP agar is solidifying at 4 °C, prepare the 5 μM treatment concentration of H2O2 in serum-free media.
    • Prepare a total of 20 mL of H2O2 in serum-free media.
    • The stock concentration of H2O2 is 100 mM.
    • Be sure to prepare with serum-free media as the diluent because the serum in media will inactivate H2O2!
  2. Retrieve your CometChips from the 4 °C and carefully transfer each into a separate container.
  3. Add 20 μL of either media or media + 5 μM H2O2 to the appropriate container.
  4. Carefully transport your CometChip to the 4 °C cooler and incubate for 30 min.
  5. Retrieve the CometChip from the cooler.
  6. Immediately transfer the CometChip to alkaline lysis buffer to stop the cellular response.
  7. To ensure cell lysis is complete, incubate the CometChip macrowells in the 4 °C cooler for 24 hrs.

Electrophorese CometChip macrowells

Due to the timing of cell lysis, the Instructors will complete the below steps.

To ensure the steps required for preparing a CometChip are clear, the Instructor will provide a live demonstration of this process. The steps are detailed below so you can follow along!

  1. Remove your CometChip from the alkaline lysis buffer and wash in 1X PBS.
  2. Use a kimwipe to dry the GelBond side.
  3. Carefully move your CometChip to the gel electrophoresis station in the 4 °C cold room.
  4. Place your CometChip on the raised center region of an electrophoresis box.
    • Double-sided tape was applied to the gel electrophoresis box. Be sure you lay your CometChip on the tape strips and lightly press down with a pipet tip to ensure it is secure.
  5. Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your CometChip.
  6. Leave the CometChip in the alkaline electrophoresis buffer (aka unwinding buffer) for 45 minutes.
  7. To separate the damaged DNA into 'comets', electrophorese for 40 min (at 16 V, or 1 V/cm).
    • It is important that the electrophoresis occur at 300 mA. To maintain the appropriate current, the volume of electrophoresis buffer may need to be adjusted. The teaching faculty will assist you in adding/removing electrophoresis buffer such that this value is reached.
  8. Carefully remove your CometChip from the electrophoresis box and place it in a dish.
  9. Obtain an aliquot of neutralization buffer from the front laboratory bench.
  10. Wash your CometChip by adding enough neutralization buffer to cover (~10-12 mL) and incubate for 5 min at room temperature.
    • Repeat this step for a total of 3 washes.
  11. Add the SYBR gold DNA stain to your CometChip and carefully move it to the 4 °C cooler.
    • Stain CometChip overnight.

Part 3: Draft Data summary slide for γH2AX results

To get a headstart and further feedback from the Instructors, you will draft the slide that will present the γH2AX data for your Data summary today in class. With your partner, use the template below, the Instructor comments from your previous homework assignments, and the helpful hints from the Comm Lab workshop to craft a data slide with the required elements.

Individually you and your laboratory partner each crafted figures (with a title and caption) using the γH2AX data. In this exercise you will come together to decide how to best present these data for the Data summary!

In addition to the data you analyzed for your experiment, you will also include data from a pilot experiment completed by the Instructors. Plot the values for the Instructor data (linked here) and review the raw images (linked here), then use the quantitative and qualitative data to craft a Panel B for your γH2AX figure.

Fa16 M1D7 data slide template format v2.png

Reagents list

  • agar, low melting point (from Invitrogen)
  • phosphate buffered saline (PBS) (from VWR)
  • Dulbecco's phosphate buffered saline (DPBS) (from Sigma)
  • alkaline lysis solution: 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% Trition-X
  • alkaline electrophoresis solution: 0.3 M NaOH, 1 mM Na2EDTA, pH 13.5 (from Sigma)
  • neutralization buffer: 0.4 M Tris, pH 7.5 (from Sigma)
  • SYBR gold DNA stain (from ThermoFisher)

Navigation links

Next day: Image and analyze data for CometChip assay

Previous day: Complete data analysis for γH2AX experiment