Technique: Chemotaxis Revisited – the Boyden Chamber Assay

Chemotaxis, introduced in my post on January 26, is the process by which cells migrate along a gradient of growth factor. Researchers study chemotaxis for many different reasons. First, there’s the biological study of how these cells move. Then, there’s the study of why these cells move. In my post from September 28, I describe how random movement of cells in response to an even distribution of growth factor can be measured. However, the next step is to measure whether this response can be seen in a directional manner in response to a gradient.

The easiest method, perhaps, for studying chemotaxis is the Boyden Chamber assay. The concept of the Boyden Chamber assay is straightforward. Known as a transwell system, a smaller well is suspended over the main well of a tissue culture plate. However, the bottom of this insert is not solid plastic, put a permeable membrane. Cells are added to the top of this membrane, in solution, and their migration across the membrane to the other side in response to stimuli can be measured simply by counting the cells that have passed through. A great product for this are the FluoroblokTM inserts from BD Biosciences. Cells can be fluorescently labeled, and light from cells on the top side of the membrane won’t shine through to the bottom. The layout of a basic Boyden Chamber Assay is shown (blue dots are cells, green dots are stimuli):

boyden blog

The Boyden Chamber Assay may be simple, but requires a high level of finesse. The first main concern is the use of proper controls. Not only are negative and positive controls important, but also we need to measure the directionality of response to control for random movement. I.e., although, for example, growth factor might be added to the bottom well, and cells to the top, we need to show that the a gradient is actually maintained and that it is not simply a case of growth factor being evenly distributed on either side of the membrane (equilibrium) and cell movement increased in general.

Also, there are several steps in which human inaccuracy can lead to deviation between wells, ultimately resulting in lack of statistical significance because of too much variability. The assay is indeed incredibly sensitive to both the number of cells and the amount of stimulus, both of which tend to be very low, meaning that everything must be incredibly accurate. The first year of my PhD I conducted a gigantic Boyden Chamber assay, but got no significant results, just trends, because the error bars were too large.

The third part of the assay which requires delicacy is the counting of the cells. As I mentioned, the cells are fluorescently stained. However, in order to calculate fluorescence, because the desired cells are on the bottom of the membrane, in order to use spectrophotometry, you’d have to have a bottom plate reader. Our spectrophotometer could not be reprogrammed to do this (although in theory it had the ability). This meant that the membrane had to be cut off and mounted upright before being imaged. The cutting of the membrane is tricky, because it becomes easy to crease, which can lead to problems when trying to mount it flat (it’s very difficult to image a crinkled sample due to focus issues).

The protocol for Boyden Chamber Assays:

  1. Trypsin cells, centrifuge, resuspend in desired medium at desired density
    1. Make sure this cell suspension is evenly mixed
    2. Place cells in the top of the transwell
    3. Place desired medium in the bottom chamber
      1. Make sure the volume of the bottom corresponds to the top volume so no fluid pressure is exerted across the membrane (manufacturer’s recommendations)
      2. Incubate for the desired amount of time (good idea to perform a time-course with controls as a preliminary study)
      3. Gently aspirate medium on top and bottom, wash 3x with PBS
      4. Incubate 15 minutes in 4% PFA in PBS in order to fix the cells
      5. Wash 3x with PBS
      6. Incubate with 1:10000 DAPI in TBS with 0.1% Tween for 5 minutes at 37°C.
      7. Wash 3x with PBS
      8. Carefully cut out membranes and place, bottom-up on slides
        1. Mount with fluorescence-stabilizing hard-set mounting medium
        2. Pressing down to eliminate any air bubbles and make sure membrane is flat
        3. Image at 4x  magnification
        4. Use ImageJ to increase brightness/contrast to max, and convert to binary for quantification
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How would you like your feedback today?

I have 6 supervisors. So far, I have received draft edits from 3 of them:

  • Supervisor 1: read the thesis Intro, then reread the whole thing at a later draft version
  • Supervisor 2: has read everything but the conclusion, but can’t seem to stomach reading 5 more pages
  • Supervisor 3: has read the whole thing through, and reread the entire second half
  • Supervisor 4: read 2 pages
  • Supervisors 5 and 6 have not read anything as far as I know

And my mother was the first person to proofread the entire thesis.

So far I have edits in a variety of media:

  • Supervisor 1: prints out the drafts, writes on them with red pen, and gives them to his secretary, from whom I pick them up
  • Supervisor 2: has sent me “track changes” documents
  • Supervisor 3: prints out the drafts, marks them up, scans them, and puts PDFs in a shared folder

And my mother sent me track changes.

So far the comment highlights have included:

  • “I’m sorry, it’s really difficult when experiments don’t work out. (my mother)”
  • “This looks weird.”
  • “This is almost boring.”
  • “Surely you didn’t perfuse the whole mouse?!”
  • And “too verbose”.

I’m currently on draft #6. I haven’t yet labeled any documents as “final” yet, but I do have several “final figures” folders. I love  this comic by PhDComics, but it doesn’t quite show the enormity of having two different computers open, one with edited documents, and one with the original document, constantly scrolling through and tweaking the thesis.

 

Picture1

Ten Hundred Words of Science Challenge

I was recently told about the amazing Ten Hundred Words of Science challenge. The challenge is really simple. Try to explain what your scientific project entails, using only the ten hundred (thousand) most commonly used words in the English language.

That sounds easy enough, until you try to use the text editor, and realize that out of the paragraph above there are many many words that would not be allowed: recently, challenge, scientific, project, entails, thousand, commonly, English, language… and SCIENCE. Science is not one of the top 1000 words in English!

This is my project description I ended up writing:

My college paper is about a fix for when a human breaks a leg or other body part. Many people in the world are working on such things. The best way to do this is to come up with a thing, that turns into part of the leg when it is put into the person, instead of trying to make a leg outside of the person and then put it in. To do this we need to focus on having good blood roads quickly, and then good leg can form. We also need to give our pretend leg everything it needs so that it can become real leg over time.

Thank goodness leg and blood was allowed! Neither bone, nor transplant, nor vessels, were allowed, which are very key words in my thesis. In the current thesis draft I mention the word “bone” over 500 times.Above, “college paper” translates into PhD thesis, leg = bone, and blood roads = blood vessels. Oh, and “thing” and “pretend leg” translate to “transplant”.

Are you up for the challenge? Scientific or not, I dare you to attempt to explain what you do in only ten hundred words! The tool can be found here:

The Up-Goer Five Text Editor

And the website to view other people’s scientific projects is here:

Ten Hundred Words of Science