Difference between revisions of "20.109(F19):Complete gamma-H2AX assay and prepare CometChip (Day3)"

From Course Wiki
Jump to: navigation, search
(Part 2: Design experiment to optimize CometChip loading)
(Reagents list)
 
Line 116: Line 116:
 
*blocking buffer: 1% bovine serum albumin (BSA) in TBS (from Sigma)
 
*blocking buffer: 1% bovine serum albumin (BSA) in TBS (from Sigma)
 
*1:1000 primary antibody to γH2AX, mouse (from Millipore)
 
*1:1000 primary antibody to γH2AX, mouse (from Millipore)
*1:200 Alexa Fluor 488 goat anti-mouse IgG (from ThermoFisher)
+
*1:200 Alexa Fluor 594 goat anti-mouse IgG (from ThermoFisher)
 
*Click-iT EdU imaging kit (from Invitrogen)
 
*Click-iT EdU imaging kit (from Invitrogen)
 
**1X Click-iT reaction buffer
 
**1X Click-iT reaction buffer

Latest revision as of 20:47, 25 September 2019

20.109(F19): Laboratory Fundamentals of Biological Engineering

Fa19 20109 Banner image.png

Fall 2019 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Measuring genomic instability        2. Modulating metabolism        3. Testing chemical probes              


Introduction

You will use commercially available antibodies to identify γH2AX foci in your experiment. The ability to bind specific proteins using antibodies, or immunoglobulins, is critical in immuno-fluorescence labeling analysis. Antibodies are typically 'raised' in mammalian hosts. Most commonly mice, rabbits, and goats are used, but antibodies can also be raised in sheep, chickens, rats, and even humans. The protein used to raise an antibody is called the antigen and the portion of the antigen that is recognized by an antibody is called the epitope. Some antibodies are monoclonal, or more appropriately “monospecific,” and recognize one epitope, while other antibodies, called polyclonal antibodies, are in fact antibody pools that recognize multiple epitopes. Antibodies can be raised not only to detect specific amino acid sequences, but also post-translational modifications and/or secondary structure. Therefore, antibodies can be used to distinguish between modified (for example, phosphorylated or glycoslyated proteins) and unmodified protein.

Monoclonal antibodies overcome many limitations of polyclonal pools in that they are specific to a particular epitope and can be produced in unlimited quantities. However, more time is required to establish these antibody-producing cells, called hybridomas, and it is a more expensive endeavor. In this process, normal antibody-producing B cells are fused with immortalized B cells, derived from myelomas, by chemical treatment with a limited efficiency. To select only heterogeneously fused cells, the cultures are maintained in medium in which myeloma cells alone cannot survive (often HAT medium). Normal B cells will naturally die over time with no intervention, so ultimately only the fused cells, called hybridomas, remain. A fused cell with two nuclei can be resolved into a stable cell line after mitosis.

Generating monoclonal antibodies.


To raise polyclonal antibodies, the antigen of interest is first purified and then injected into an animal. To elicit and enhance the animal’s immunogenic response, the antigen is often injected multiple times over several weeks in the presence of an immune-boosting compound called adjuvant. After some time, usually 4 to 8 weeks, samples of the animal’s blood are collected and the cellular fraction is removed by centrifugation. What is left, called the serum, can then be tested in the lab for the presence of specific antibodies. Even the very best antisera have no more than 10% of their antibodies directed against a particular antigen. The quality of any antiserum is judged by the purity (that it has few other antibodies), the specificity (that it recognizes the antigen and not other spurious proteins) and the concentration (sometimes called titer). Animals with strong responses to an antigen can be boosted with the antigen and then bled many times, so large volumes of antisera can be produced. However animals have limited life-spans and even the largest volumes of antiserum will eventually run out, requiring a new animal. The purity, specificity and titer of the new antiserum will likely differ from those of the first batch. High titer antisera against bacterial and viral proteins can be particularly precious since these antibodies are difficult to raise; most animals have seen these immunogens before and therefore don’t mount a major immune response when immunized. Antibodies against toxic proteins are also challenging to produce if they make the animals sick.

Generating polyclonal antibodies.


In your experiment, you will use a primary antibody to bind the γH2AX foci. Then a secondary antibody will be used that is specific to the conserved region of the primary antibody. The use of secondary antibodies allows researchers to tag the primary antibody. In our assay, the tag is a 488 nm fluorescent dye that will enable us to visualize double-strand breaks via microscopy. As a reminder, during the last laboratory session you treated cells with MMS and As for the H2AX assay. Since then, the teaching faculty fixed all the wells with paraformaldehyde. Your task for today is to permeabilize the cells, which will enable the antibodies to enter the cells and bind γH2AX.

Protocols

Part 1: Begin antibody staining for γH2AX assay

Immunofluorescence staining chamber

At this point in the assay sterility is no longer a concern and you will complete the following steps in the main laboratory at your bench.

  1. Obtain your 12-well plates from the front laboratory bench.
  2. Gather an aliquot of 1 X TBS from the front laboratory bench.
    • Prepare 1.2 mL solution of 0.2% Triton X-100 (v/v) in 1X TBS in a micro centrifuge tube. Triton stock (10%) is at the front laboratory bench.
    • Prepare 2.5 mL solution of 1% BSA (v/v) in 1X TBS in 15ml conical tube. The 10% BSA stock is at the front bench.
      • One of the preparations will be the blocking solution used in Step #8 and the other preparation will be used in Step #9 for the primary antibody solution.
  3. Obtain a staining chamber from the front bench and add a damp paper towel to each side of the parafilm. Label parafilm with experimental details.
  4. Obtain a fine gauge (26 3/8) needle and a pair of tweezers from the front laboratory bench.
    • Carefully press the tip of the needle against the benchtop to bend it into a right angle such that the beveled side of the needle is the interior angle.
  5. Use the 'hook' created with the needle to lift the coverslip from the bottom of the well, then use the tweezers to 'catch' the coverslip.
    • Practice plates with coverslips will be available at the front laboratory bench.
  6. When you are confident with your ability to retrieve the coverslips from the wells, move one coverslip from each condition from your 12-well plates to the staining chamber. Cell-side UP!
    • The cell-side of the coverslip is the side that was facing up in the well of the 12-well plate.
  7. Quickly permeabilize the cells by adding 150 μL of the 0.2% Triton X-100 / TBS solution to each coverslip and incubate for 10 min at room temperature.
  8. Aspirate the 0.2% Triton X-100 / TBS solution, then prepare for the Click-iT reaction by washing the cells.
    • Add 150 uL of TBS to each coverslip.
    • Place a clean P200 pipet tip on the aspirator and carefully remove the TBS.
    • Repeat a total of 2 times.
  9. Add 0.2 mL of Click-iT reaction mix to each coverslip.
    • The Click-iT reaction mix was prepared by combining: 2.2 mL Click-iT reaction buffer, 100 μL CuSO4, 6 μL Alexa Fluor azide, and 250 μL reaction buffer additive.
  10. Cover the plate with foil to protect from light and incubate for 30 min at room temperature.
  11. Aspirate the Click-iT mix and wash the cells as done in Step #8.
  12. Add 150 μL of BSA blocking solution to each coverslip, then incubate for 60 min at room temperature.
  13. With 15 min remaining of the blocking solution incubation, prepare the primary antibody.
    • Dilute the mouse anti-γH2AX antibody 1:1000 in the 1.2 mL aliquot of BSA blocking solution.
  14. Aspirate the block solution and add 150 μL of the diluted primary antibody solution to each coverslip before moving the next. Do not let the coverslips dry!
  15. Carefully move your staining chambers to the 4 °C cooler.

Your samples will incubate at 4 °C in the primary antibody solution for ~48 h. The teaching faculty will replace the primary antibody solution with the secondary antibody solution, Alexa Fluor 594 goat anti-mouse diluted 1:200 in blocking solution, 1 h prior to the next laboratory session.

Part 2: Design experiment to optimize CometChip loading

The experiment you design will test a variable associated with loading cells into the microwells of your CometChip. The variable in this experiment is cell number. Specifically, how many cells should be added to each macrowell to ensure the majority of the microwells are loaded? In addition, how many cells should be loaded into each microwell?

On your CometChip there is space for you to complete this experiment using three conditions. Your experiment will address the question above and the conditions will provide data that will, hopefully, answer your research question.

CometChip schematic for loading variables experiments.

Experiment: Determine the number of cells needed to completely load the microwells of your CometChip

Distinction between (A) 'macrowell' and (B) 'microwell' for the CometChip assay.
This experiment has two questions that should be considered as you discuss the conditions you will test with your partner. Before moving on to these questions, it is important to differentiate between the terms 'macrowell' and 'microwell' for your experiments. A bottomless 96-well plate is placed on top of the agarose CometChip to create the macrowells for the CometChip assay (panel A). This enables researchers to control which cells are exposed to which treatment. The microwells were stamped into the agarose when you made your CometChip (panel B). Within each well are ~ 300 microwells, which are ~40 μm in diameter and 40 μm in depth.
  1. How many cells should be loaded into each microwell?
    • The goal is to use as few cells as possible. This will limit waste, preserve resources, and reduce cost!
    • Consider the amount of DNA that is carried by a single mammalian cell and the detection limit provided by the SYBR gold DNA stain that will be used in your experiments. Also, use the data you collected during M1D1 Part 3 to determine how many cells can fit into a single microwell based on the dimensions provided above.
    • Be sure to include any calculations or thoughts in your laboratory notebook!
    • When you know how many cells you want to load into each microwell, move on to the next question.
  2. How many cells should be added to each well such that the desired number of cells are loaded into the microwells?
    • 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 as you consider this question.
  3. Now that you have an idea as to the number of cells that are ideal for loading, consider the conditions you will use in your experiment.
    • Your team will choose two 'cell number' conditions for your experiment.
    • You will be provided with cell suspensions at 500K cells / ml for the loading experiment. Calculate the volume of cells that you will need for each of your conditions keeping in mind that you must add at least 50 μL and no more than 350 μL.
  4. Again, all information concerning your experimental design choices should be recorded in your laboratory notebook!
  5. Alert the teaching faculty when you are ready to load your cells for the cell loading experiment.

Part 3: Load CometChip

  1. Retrieve your CometChip from the 4 °C cooler.
    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.
  2. Remove your CometChip from the 1x PBS and place it, gelbond side down, on the glass plate.
  3. 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.
    • 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.
  4. 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 the white wells and the grey wells should remain empty.
  5. Add 50 μL of 1x PBS to the Condition A wells.
  6. Add the appropriate volume of your cell suspension (calculated in Part #2) to the Condition B and C wells.
  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.
    • Alert the teaching faculty at this point! The wash step can be very temperamental and it is best to see a demonstration!
    • 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 Condition C is at the bottom.
      • To waterfall the 1x PBS, 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. Read through Steps #11-14 before continuing with the procedure.
  10. 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.
  11. Using the P1000, pipet up 1000 μL of molten agarose from the tube.
  12. 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!
  13. 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.
  14. Use the microscope in the main laboratory to image your CometChip. You will use these images to determine: 1. the number of microwells that are loaded / total number of microwells in the frame and 2. the number of cells / microwell.

Reagents list

γH2AX

  • permeabilization buffer: 0.2% Triton in Tris buffer saline (TBS) (from Invitrogen)
  • blocking buffer: 1% bovine serum albumin (BSA) in TBS (from Sigma)
  • 1:1000 primary antibody to γH2AX, mouse (from Millipore)
  • 1:200 Alexa Fluor 594 goat anti-mouse IgG (from ThermoFisher)
  • Click-iT EdU imaging kit (from Invitrogen)
    • 1X Click-iT reaction buffer
    • copper sulfate (CuSO4)
    • Alexa Fluor azide
    • Reaction buffer additive
  • Mounting media ProLong gold with DAPI (from ThermoFisher)

CometChip

  • agar, low melting point (from Invitrogen)
  • phosphate buffered saline (PBS) (from VWR)

Navigation links

Next day: Perform immunofluorescence imaging for repair foci and expose cell for high-throughput genome damage assay

Previous day: Perform cell exposures for repair foci experiment and perform high-throughput genome damage pilot