20.109(F19):Load cells into CometChip and apply treatments (Day4)

Introduction

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.

CometChip assay results.

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.

Protcols

Part 1: Load CometChip

You will use a CometChip and cells prepared for you by the Instructors to load the CometChip in the main laboratory. It is important that you consider the following details before loading for your experiment.

1. The density of your cell suspension (500K cells / mL) will determine the volume you load into each macrowell of the CometChip.
   • Calculate the volume of cell suspension that you will need to load such that 25K cells (or the number your group determined was optimal) are added to each macrowell. Check your math with the Instructor before moving forward.
2. Retrieve a CometChip from the front laboratory bench.
   

   

   CometChip diagram showing well alignment.
   • You will also need to gather one glass plate, one 96-well bottomless plate, and four 1.5" binder clips from the front bench.
3. Remove your CometChip from the 1x PBS and place it, gelbond side down, on the glass plate.
4. Press the 96-well bottomless plate upside-down onto the CometChip so that the wells line up with your labeling as shown in the diagram on the right!
   • The total size of the CometChip should be 5 wells across and six wells down. You should have extra space / wells around the perimeter.
   • 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.
5. 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 below.
     

     

     Binder clip placement for CometChip sandwich.
   • You will load into 30 wells total; 5 across by 6 down according to the map on the right!
6. Add the appropriate volume of your cell suspension (calculated in Step #1) to each of the macrowells.
7. Cover the top of your CometChip with plastic wrap then incubate in the 37 °C incubator in the main laboratory for 15 min.
8. 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.
9. 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.
10. Using the P1000, pipet up 1000 μL of molten agarose from the bottle.
11. 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!
12. 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.
13. Use the microscope in the main laboratory to ensure you have cells loaded into your CometChip.

Part 2: Treat cells for DNA damage experiment

Apply MMS treatment

1. While the LMP agar is solidifying at 4 °C, calculate the dilution of MMS that you will use for your experiment.
   • The MMS stock solution is at a 12 mM concentration.
   • You will add 100 μL per well to induce DNA damage.
   • Calculate the dilution for a dose of 0.4 μM.
2. Obtain an aliquot of MMS and prepare the dose that you calculated in Step #1 using DMEM media without supplements as the diluent.
   • Be very careful with MMS and work on the absorbent paper at all times!
     

     

     DNA damage experiment plate map.
3. Retrieve your CometChip from the 4 °C and carefully replace the bottomless 96-well plate to recreate the wells that you used for cell loading.
   • Secure the bottomless 96-well plate with binder clips as before.
4. Add 100 μL of either DMEM or DMEM + 0.4 mM MMS to the appropriate wells according to the plate map on the right.
5. Carefully transport your CometChip to the 37 °C incubator for 1 hour.

Apply As treatment

1. During the MMS treatment, calculate the dilutions of As that you will use.
   • The As stock is at a 100 mM concentration.
   • You will add 100 μL per well to induce DNA damage.
   • Calculate dilutions that result in doses of 0, 20, 40, 60, and 80 μM.
2. Confirm your match with the teaching faculty, then prepare the dilutions with ~15 min remaining in the MMS treatment.
   • Obtain an aliquot of As and prepare the dose that you calculated in Step #1 using DMEM media without supplements as the diluent.
   • Prepare the doses that you calculated in a 12-well reservoir as discussed in prelab.
   • As before, be very careful with As and work on the absorbent paper at all times!
3. Retrieve your CometChip from the 37 °C incubator and use the P200 set at ~120 μL to remove the DMEM or DMEM + 0.4 mM MMS.
4. Using a multi-channel pipetman, add the appropriate treatment doses of As to each well of your CometChip.
   • The 0 μM treatment should be added to the wells at the far left and the 80 μM treatment should be added to the wells at the far right of the CometChip.
5. Carefully transport your CometChip to the 37 °C incubator for 2 hours.
6. Rinse your CometChip using 1x PBS into the same dish used during the cell loading procedure.
   • Complete a total of 3 washes.

Part 3: Complete cell lysis

1. Use the same dish your CometChip was stored in at the start of lab and a 30 mL aliquot of alkaline lysis buffer from the front laboratory bench.
2. Carefully move your CometChip into the dish.
3. Add the aliquot of alkaline lysis solution by slowly pouring it into a corner of the dish rather than directly over the CometChip.
   • Be sure the CometChip is completely submerged and not floating in the dish.
4. Label the dish with your team and section information.
5. Move your dish into the 4 °C cooler.

The teaching faculty will transfer your CometChip from the alkaline lysis solution into 1X PBS at ~24 hr.

Reagents list

• agar, low melting point (from Invitrogen)
• phosphate buffered saline (PBS) (from VWR)
• Dulbecco's Modified Eagle's Medium (DMEM) (from Gibco), without supplements
• methyl methanosulfonate (MMS) (from Sigma)
• arsenite (As) (from Sigma)
• alkaline lysis solution: 2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris, 1% Trition-X

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