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

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.



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

Technique: Chemotaxis concepts – Why did the chicken cross the road?

Why did the chicken cross the road? Well, frankly, who knows what induced the chicken to cross the road. Generally, we can assume that the chicken had a reason. Arguably everything we do in everyday life we do for a reason – whether voluntary or involuntary. Like most other creatures in the world, we respond to our environments.

If you show a dog a bone, and then place that on the far side of the room, the dog will run over to get it. But the stimulus doesn’t have to be food. If a person enters the room, a dog will typically rush over to greet it – though whether that greeting is friendly or threatening depends on many factors.  Lack of movement can also be due to the environment that we sense around us. For example, when I often wake up, I am tempted not to get out of bed, because it Is incredibly comfortable where I am, and, for the time being, there is no specific reason to move.  Therefore:

Movement = response to factors signalling that another place is desirable and/or lack of factors signalling that the current location is desirable

Lack of movement = response to factors signalling that the current location is desirable and/or lack of factors signalling another place is desirable

Like whole organisms, cells behave in response to the environments they sense around them. For example, consider cells in the circulation. While initially one thinks of these cells as being simply pushed around by the physical pressures exerted by fluid dynamics, these cells are actually constantly sensing their environments and can respond by clotting, leaving the leaving the blood system and moving into surrounding tissues, or continuing circulating. In contrast, think of skin cells. These cells stay in one place, because they are surrounded by similar cells and supporting layers of cells that provide the signals to stay in place. However, in response to injury, e.g. a paper-cut, the cells quickly change their entire behaviour and can both move into the wound site as well as signal to the blood and immunological cell types necessary for repair.

Outside of the body, in the laboratory setting, it’s easy to study the effect of each movement signal, also known as a chemotactic factor (chemotaxis meaning movement in response to a chemical signal) because we can add the factors in individually and in combinations to observe their exact effects. In this blog I have already discussed a way to measure random cell movement in response to growth factors (September 28: Calculating Cell Velocity). In my next post I will discuss the Boyden Chamber assay for studying directional cell movement in response to factors.


And now, my body is telling me I need to move to a location that provides caffeine. I’m off to get some coffee and get back to working on my thesis.

Thesis Writing: The Middle Chapters

The middle chapters of the thesis include the Methods, Results, and Discussion sections. These are, to a certain extent, the hardest chapters of the thesis to write. Unlike, for example, the introduction, it’s not simply a questions of laying out your ideas, looking up what other people have done that is relevant, and discussing their work. The Methods section, I grant you, is very straightforward as long as you have documented what you have done along the way – you, after all, should know what experiments you did. The Results section is slightly trickier, in that you need to figure out how to logically and best present your findings in a way that conclusively demonstrates your point without overwhelming the examiners with extraneous data or confusing them. The Discussion section is by far the most difficult as you must evaluate your data and explain how it fits in with the overall aims of the project.

So far, in my posts on thesis writing, I’ve discussed the general format of the thesis (August 27) and my current efforts with the results section (November 17). Particularly, in the August 27th post, I stressed that the PhD is not simply a long paper. That said, there are countries in the world, e.g. Sweden, Finland, the Netherlands, and Australia (to name a few) that will accept several papers written by the student (and published in peer-reviewed journals) stapled together in lieu of an actual thesis.   If thesis writing were like just writing a long paper, there would be the following sections: Introduction, Materials and Methods, Results, Discussion, and Conclusions. However, many people would argue that while a thesis needs an Introduction section as well as a Conclusions and Future Work section, the middle chapters should not be so strictly organized. Rather, the belief of having several middle chapters based on various themes or questions pertaining  to the overall project which each contain subsections of Materials, Results, and Discussion, is becoming increasingly prevalent within the scientific academic community.   In fact, a friend of mine who recently defended in Oxford had organized his thesis as if a long paper and was told that for his final version the only alterations desired were that he cut and paste his sections in the latter method to tell a better story.

Part of the reason why this approach of having self-contained chapters that each contains several parts, is that it lends a better flow to the thesis. Because theses tend to be so long, it becomes difficult for an examiner (one dare not hope that anyone other than the examiner will actually attempt to read the full document) to read each section in its entirety and keep it fresh in their mind as they go on to the next section.  Indeed, if organized thusly, the method, results, and discussion of a particular method could be spaced out with 20 or more pages in between them – hardly allowing for the formation of a sense of direction. However, by splitting the middle section of the thesis into topical chapters, this allows for the examiner to focus on a smaller topic at each time, with a sense of how each experiment was performed, what the results are, and how to interpret these results in relation to other experiments which also answered the same question. Then, with an idea of how each topic was addressed, the parts can be brought together in the final Conclusion section to show the larger picture.

The simplest way, perhaps, of explaining this concept is to think of the difference between a nursery, and a garden.  If the Methods, Results, and Discussion sections were three types of flowers, then the more traditional method would be akin to a nursery; each section would be taken care of, but separately and in neat little rows. However, the newer method would see that although a garden contains these three types of flowers, in contains them in several beds which contain clusters of each type, so that the flowers can be seen in relation to each other, followed by the design and layout of the beds.

Middle chapters

Fatigue related to outdoor darkness

Living in England in the heart of winter can make for quite a dark time, literally. For a number of months the sun does not rise until after 8 AM and sets before 4PM.  Even  people who have lived in the country all of their lives will tell you that this has the effect of making it seem much later than it is in the evenings. There is no denying that the environmental darkness that we experience  an effect on our psychological state, and possibly our physical state as well. I recently experienced this first-hand after spending almost 2 weeks in South Africa on holiday, where the sun rose before 6 and set after 8pm. Upon my return to Oxford I immediately lost over 6 hours of sunlight with no jetlag (time difference is only 2 hours). I find myself asking for dinner around 4:30 in the afternoon because it looks like dinner-time outside, and simply getting more tired in the evenings.

But what does science have to say about all this? The science of how our bodies regulate themselves on a daily cycle is known as the study of circadian rhythms. And indeed, the concept of circadian rhythms is not just psychological, with changes in body temperature, gastrointestinal, endocrine, and respiratory functions affected. Metabolism is also affected (though whether that or my brain is more at work with my 4:30 desire for dinner is debatable). Popular topics of study with circadian rhythm scientists are jet lag, shift work (i.e. if you have to work nightly shifts), and seasonal affective disorder (SAD). All of these are instances where our bodies experience changes in environment from what is considered the norm.

Jet lag is easily explainable through the study of circadian rhythms. By shifting time zones, your body immediately senses the new environmental conditions, and gradually your rhythms shift to become in line with this. However, as the cycles take time to align, this is what results in the feelings of jet lag – with changes in behaviour and performance observed. Working the night-time shift is more difficult than switching time zones because it involves using the body against some of the environmental cues (sunlight) that normally govern its functions. In these instances other environmental cues, such as social interactions, become more important to try and shift the cycle. However, studies have shown that individuals who work these shifts do exhibit behavioural problems and social isolation, compared to those working normal shifts.

So how does this all play out with respect to the English winter? Well, in winter we get a conflicting set of cues. True, the time in which we experience sunlight diminishes, but our work schedules and social interactions with others stay on the same time scale, in effect, helping out where the environmental cues are now absent. The hypothesis is that our circadian rhythms are phase-delayed in winter as contrasted to summer, but this has not been conclusively demonstrated. Interestingly, much research has been done with special bright light therapies, in order to reduce the symptoms of SAD. While these therapies do generally improve symptoms, they somehow do not appear to be effected by when they are administered. This confuses scientists, who suspect that it might be having a “sophisticated placebo or expectation response” effect.

The general outlook of the English is to simply accept the situation as it is, and take the occasional winter holiday to Spain or Mallorca. But perhaps the finding that night-shift workers deal with the situation in better in older age, due to advanced coping mechanisms, is the most illuminating.

Disorders of the sleep-wake cycle in adults

On New Year’s Resolutions

Do New Year’s resolutions work? Do they set us up for disappointment? Are they a marketing ploy to increase sales, particularly of fitness equipment and gym memberships, at the beginning of every year? Or can they actually be helpful, an honest effort and impetus, however, brief, to genuinely improve our lives?

Different studies over the years have shown high levels of initial success, e.g. 1 week, diminishing more rapidly by a month, and with limited success after one and two years. In general, it appears, individuals fail more frequently when they make weight resolutions versus non-weight related resolutions, e.g. smoking or drinking.

An interesting study, published in 2002, aimed to not just measure the rates of resolution success, but also the driving forces behind it, and to directly answer whether resolution actually effected change as contrasted to individuals contemplating change. The study indicates that its success rates may be slightly inflated due to the nature of self-reporting, with less successful applicants declining follow-up interviews or making up more successful results, and with some of the participants being motivated to stick to their resolutions because they know they will have follow-up interviews. However, it is important to note several things from the study. First, that 40% of American adults make New Year’s Resolutions every year. Second, that non-successful resolvers are likely to make the same resolution the following years until some degree of success has been achieved. And third, that resolving is not in vain and that most participants do achieve initial success, even if this is not particularly long lasting. As the authors point out, the rate of success should be compared to non-resolvers, i.e. people who don’t make a conscious effort to improve at all.

Most importantly, and surprisingly, once the initial resolution had been made, desire to change had no correlation with resolution success. Instead, success was found by participants who used techniques of “self-liberation, stimulus control reinforcement management, positive thinking, and avoidance strategies”, whereas those who were unsuccessful were characterized by “self-reevaluation, wishful thnking, self-blame, and minimized threat”. That is, those who were more successful better managed and kept on top of the situation and goals, whereas those who were unsuccessful were more likely to get frustrated, blame themselves, and/or talk themselves out of the need to change.

Thus, it’s better to try out a New Year’s resolution than not at all. And above all, stay positive. What’s your New Year’s resolution?

Auld Lang Syne: Success Predictors, Change Processes, and Self-Reported Outcomes of New Year’s Resolvers and Nonresolvers