Difference between revisions of "20.109(F18):Design cell loading optimization experiment and discuss genomic instability experiment (Day2)"

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(Part 3: Load CometChip)
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You will use the cells you prepared in the tissue culture hood to load the CometChip in the main laboratory.  It is important that you consider the following details before entering the tissue culture room.
 
You will use the cells you prepared in the tissue culture hood to load the CometChip in the main laboratory.  It is important that you consider the following details before entering the tissue culture room.
 
#The density of your cell suspensions will determine the volumes you load in the CometChip for Conditions B and C.
 
#The density of your cell suspensions will determine the volumes you load in the CometChip for Conditions B and C.
#*Calculate the volume of this suspension that you will need to load for Conditions B and C.  If the calculated volumes are less than 50 μL or greater than 400 μL, consult the teaching faculty with how to move forward.
+
#*Calculate the volume of this suspension that you will need to load for Conditions B and C.  If the calculated volumes are less than 50 μL or greater than 350 μL, consult the teaching faculty with how to move forward.
 
#Retrieve your CometChip from the 4 °C cooler.[[Image:Fa18 M1D2 cell loading map v2.png|thumb|350px|right|CometChip diagram showing well alignment.]]
 
#Retrieve your CometChip from the 4 °C cooler.[[Image:Fa18 M1D2 cell loading map v2.png|thumb|350px|right|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.
 
#*You will also need to gather one glass plate, one 96-well bottomless plate, and four 1.5" binder clips from the front bench.

Revision as of 17:20, 14 September 2018

20.109(F18): Laboratory Fundamentals of Biological Engineering

Fa18 20109 banner image.png

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


Introduction

Today you will load cells into the CometChip you prepared during the previous class to test cell loading conditions in an effort to optimize the assay developed by the Engelward Laboratory for our experimental purposes.

Cell loading refers to the process by which the cells are added to the CometChip. Briefly, you will add media containing cells over the agarose-based CometChip, and gravity will pull the cells down and 'load' them into the CometChip. The experimental details of this process will be the basis of the experiments you design and perform during this laboratory meeting.

Experimental design refers to the process by which the details of an experiment are organized to ensure that the data collected are appropriate and answer the correct question. In an experiment, a treatment is intentionally imposed on a sample such that the outcome(s) can be observed. You should consider these points when designing an experiment:

  • Treatments should be administered in measurable levels. The level, or amount, of treatment must be conserved across samples to limit unintended variability in the results.
  • Controls should be included. The controls, or untreated samples, are a baseline to which the treated samples are compared.
    • In the negative control group, no result is expected. This treatment will lack enzyme or other important factor necessary for the result / reaction that is being tested.
    • In the positive control group, an expected result is generated to ensure that you can see what you are testing.
  • Each experiment should have only one variable. If multiple variables (e.g. treatments, conditions, etc) are included in a single experiment, the results will be inconclusive because the outcome may be attributed to any of the variables within the experiment.
  • Replicates should be included. The replicates test for technical error introduced by the researcher.

Designing a successful experiment requires time, effort, and practice. Today you will design an experiment to interrogate the best conditions for loading cells into your CometChip.

Protocols

During any downtime, work through Part #4 with your laboratory partner.

Part 1: 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 4 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.
  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 prepare your cells for the cell loading experiment.
    • Note that the tissue culture room cannot accommodate the entire class at the same time. Please be patient and complete Part 4 while you wait for an open hood.

Part 2: Prepare cells for CometChip loading

  1. Clean the tissue culture hood and prepare it with the supplies you will need.
    • Carefully read through the protocol to ensure you have everything you need at hand.
  2. Retrieve the flasks you seeded during the previous class session from the 37°C incubator.
  3. Prepare cell suspensions for use in your cell loading experiment as done previously. Briefly,
    • Examine your cell culture.
    • Aspirate the media.
    • Wash the cells with 5 mL PBS.
    • Dislodge the cells with 1 mL of trypsin.
    • Incubate the cells at 37°C for 2 minutes using a timer.
  4. Retrieve your flasks from the incubator.
    • Check your cells using the microscope to ensure they are dislodged. They should appear round and move freely. If they are still partially adhered, incubate for longer and keep track of the time. Note: Since the M059J (-DNAPKcs) cells tend to aggregate, you may consider trypsinizing them longer to help dissociate them.
    • Once the majority of the cells are detached, firmly tap the bottom to fully dislodge the cells.
    • Add 4 mL of media to the cells then triturate to break up cells that are clumped together and suspend them in the liquid.
    • Transfer the suspended cells into a labeled 15 mL conical tube.
    • Transfer 90 μL of each cell suspension from the 15 mL conical tube into a labeled eppendorf tube.
    • Save the remaining cell suspension in the 15 mL conical as this is what you will use to load your CometChip!
  5. Clean out the tissue culture hood.
  6. Determine the number of cells / mL in your cell suspension as done previously. Briefly,
    • Add 10 μL of trypan blue to the 90 μL aliquot of each cell suspension.
    • Use the hemocytometer to count the cells, then calculate the cells / mL in each suspension.
  7. If necessary, use fresh media to dilute your cells such that at least 50 μL is added into each well for your determined cell number in your experiment.

Part 3: Load CometChip

You will use the cells you prepared in the tissue culture hood to load the CometChip in the main laboratory. It is important that you consider the following details before entering the tissue culture room.

  1. The density of your cell suspensions will determine the volumes you load in the CometChip for Conditions B and C.
    • Calculate the volume of this suspension that you will need to load for Conditions B and C. If the calculated volumes are less than 50 μL or greater than 350 μL, consult the teaching faculty with how to move forward.
  2. 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.
  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.
    • 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 the white wells and the grey wells should remain empty.
  6. Add 50 μL of 1x PBS to the Condition A wells.
  7. Add the appropriate volume of your cell suspension (calculated in Step #1) to the Condition B and C wells.
  8. Cover the top of your CometChip with plastic wrap then incubate in the 37 °C incubator in the main laboratory for 15 min.
  9. 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. Try not to mix the two cell lines.
      • 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.
  10. Read through Steps #11-14 before continuing with the procedure.
  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.
  15. 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.

Part 4: Research the M059K and M059J cell lines

In this exercise you will learn more about the cell lines you are using in this module. The wild-type strain is called M059K. Wild type is used to describe the phenotype of a typical or non-mutant form of a bacterial species or cell line. The mutant strain is called M059J and does not generate a subunit of DNAPKcs, rendering the protein inactive. A great resource from which we can gather information on these cell lines is the American Tissue Type Collection (ATCC). Use the information concerning M059K and M059J to answer the following questions in your notebook:

  1. When were these fibroblast cells originally acquired?
  2. What characteristic of the cell is described by the term 'fibroblast'?
  3. Do these cells float in the culture media, or stick to the culture flask? How do you know?
  4. From what organ tissue were these cells harvested?
  5. For what types of studies are these cells a useful model system? What evidence is provided to support this?
  6. How should these cells be cited in a scientific publication?

The M059J strain was first described by Allalunis-Turner et al. in a research article found here. Use this paper to answer the following questions in your notebook:

  1. Read the introduction.
    • Why might it be useful to have model cell lines that exhibit sensitivity to radiation or drugs?
    • The purpose of an introduction is to provide the reader with information necessary for understanding the purpose and results of the paper. Is there any additional information that would be helpful to include in this introduction?
  2. Read the results.
    • Review Fig. 3. In the results section, the authors state that the "SF2 values calculated for M059J and M059K cells were 0.02 and 0.64, respectively." Read the portion of the Colony-forming assay paragraph that discusses the radiation assay in the methods section. Briefly, discuss how these data were generated. What additional information would be useful if you wanted to repeat this experiment?
    • Review Fig. 4 and 5. What do these data suggest concerning the role of DNAPKcs in double-strand break repair? Consider a possible mechanism by which the drugs are interacting with DNAPKcs.
    • Review Fig. 6. Are the results for M059J and M059K what you expect? Why or why not?
  3. Read the discussion.
    • Why is the authors' approach to acquiring cell lines from individual tumors with differing properties preferable?
    • Within the discussion authors often present caveats of their research. What do the authors note about their cell lines that should be considered by the reader?

The radiosensitivity phenotype of M059J was further explored by Lees-Miller et al. in a research article found here. Use this paper to answer the following questions in your notebook:

  1. Read paragraphs 1-4.
    • Draw a diagram that includes a DNA double-strand break, DNAPKcs, Ku70, and Ku80, depicting the relationship between the molecules. Feel free to use sources outside of the paper.
    • What is known about the function/role of DNAPKcs?
    • What do the authors suggest is the reason for the radiosensitivity phenotype of M059J cells?
  2. Read paragraphs 5-9.
    • What experiment(s) did the authors complete to test for the presence of the DNAPKcs protein? the gene that encodes DNAPKcs? the activity of DNAPKcs?
  3. Read paragraphs 10-end.
    • What do the authors suggest is the root cause of the M059J phenotype? Do the data support this interpretation? Why or why not?

Reagents list

CometChip:

  • agar, normal melting point (Invitrogen)
  • GelBond film (Lonza)
  • 1 well dish (VWR)
  • agar, low melting point (Invitrogen)
  • bottomless 96-well plates (VWR)
  • phosphate buffered saline (VWR)

Navigation links

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