Friday, February 27, 2015

Just a Small Town Girl, Living in a Snowy World

This week, I saw snow. I know snow isn't exactly uncommon (especially on the East Coast right now), but where I live, even rain is a Facebook event. And snow? That'll blow up your feeds for the entire day. I admired the snow for a few minutes, but the novelty wore off as soon as I went outside. Snow is cold; I am not. Unfortunately for me, I had to walk in this weather from the bus stop to the house. I'm not looking forward to doing this again next week. Fortunately, I had a nice warm coat so I didn't get too cold (except for my nose). I'd like to thank the parental units (again) for getting me said coat.

Weather aside, I'd like to think that this week went pretty well (in the lab, at least), although I was a bit dubious in the beginning. I'll use this post to explain more in detail what I'm doing, so bear with me (or skip until the end if you really want. I won't know the difference).

First, I got four small vials and had to clean them. To this end, I took them to the fume hood and washed them with acetone, chloroform, ethanol, and petroleum ether (oh my). Why I was trusted with these chemicals, I'll never know. But the lab is still in one piece! For now, anyways. Then, I turned around and saw this lovely sight.

"If you kill these slices, be prepared to donate your own brain cells for today's experiments."

Ah, I feel welcomed already. On a side note, I was able to see the last frog surgery on Monday. They wanted to collect oocytes for further testing of the M2 channel. It's similar to what I'm doing, but they use mRNA to make the M2 channels, while I'm artificially making liposomes with M2 channels.

Next, I add around 150 μL of lipids, which are suspended in chloroform, to each vial. After placing parafilm (how is that stuff so stretchy?) on three of the vials, I place the other vial on the rotavapor, which spins the vial while pumping a continuous stream of nitrogen gas. Eventually, the chloloform is gone, and the lipids are stuck to the end of the vial. I repeat this with the other three vials. When I'm not using the vials, I always put parafilm over them; the oxygen in the air can react with the lipids, making them unreliable in the testing phase. It'd be rather unfortunate if I labored over these lipids and liposomes for hours, only to have the liposomes be leaky during the test (although, I'm sure this'll happen eventually).

Then, I add around 180 μL of both chloroform and wildtype M2 (meaning that the M2 channel has not mutated) to two of the vials. And then I repeat the rotavapor phase, but I sit there longer, staring off into space, because the M2 channels were suspended in methanol. Methanol takes much longer to evaporate off, although adding the chloroform makes it go a bit faster for some reason. Now that I think about it, I could probably read during this time. Or I could be efficient in making more. Decisions, decisions....

After adding about 150 μL of internal buffer to each vial, I put them on the vortexer and sonicator, and then repeat this until there aren't any more lipids on the sides of the vials. Finally, I store these mixtures in the fridge. Viola! We have liposomes. And thus, my day one of this process is complete. (I know, I know. I'm sorry, this is going to be a long post.)

On day two, my fingers get a work out. I have to heat up the internal buffer, the vials, and the extruder (this sounds kind of ominous) to 57 degrees Centigrade. However, before I heat the extruder up, I have to assemble it. To do this, I wash the pieces with methanol, then place filter supports, a 0.2μ filter, and more filter supports, into the chamber. Here's what it looks like when it's all assembled with the syringes.

It's not so scary now, is it?

The heating just makes the extruding process easier (or so I'm told). In this process, I first put some internal buffer into the syringe on the right. Then, I push that syringe in, and the liquid (somewhat magically, to me at least) goes into the syringe on the left. I repeat this ten more times, so that the liquid ends in the syringe on the left. Because of this, the filter absorbs some liquid and makes extrusion easier (easier is better, trust me).

After I remove the internal buffer solution, I put all of one vial into the right syringe. Usually, I use a vial without the wildtype M2 channels first. Next, I'll push that through the filter 21 times, so, again, the mixture ends on the left, and I'll deposit that into an Eppendorf tube (such a big name for a small vial. The picture actually shows the final products after extrusion, when the mixtures are in the Eppendorf tubes.). Since I have another vial that is also a blank (that is, it has no M2 channel), I can filter that through as well. Then, I repeat this whole heating-and-assembling-extruder process (which is harder than it seems, since the filters and filter supports are so small), just so I can extrude the mixtures that have the wildtype M2 channels. (And, let me tell you, extruding liposomes with the M2 channel is a lot more difficult.) Finally, I let the liposomes sit for about a day in room temperature.

Why do we extrude the liposomes, you might ask (well, I asked, anyways). Although we can get better signals (I'll explain later) from bigger liposomes, having liposomes completely different sizes makes the signals a bit unreliable. By extruding the liposomes, they will be roughly the same, small size, so this makes it easier to replicate the experiment. 

And all of this prepwork leads up to day three! You'd think that we'd have some really awesome experiment that takes a while to run and is extremely satisfying, but that's not exactly the case. For the test, we use the pH meters I showed you in an earlier post. These pH meters don't actually read the pH, though; they read a voltage, which can then (somehow) be converted to a pH. 

First, I add 3 mL (pretty much the only measurement that isn't in μLs) of external buffer and a stir bar (small magnets that spin based on a current going through a coil of wire) to a different, smaller vial. The actual test only lasts about 10 minutes per sample. I start by adding 30 μLs of 0.1 M HCl and 30 μLs of one sample to the external buffer (and I add 30 μLs of a drug solution to two of the mixutres), then I start the pH meter. Throughout the test, I add valinomycin, CCCP, 0.001 M HCl (twice), valinomycin, and CCCP. I'll explain what these molecules do and why we add them in a later post.

Finally (for real this time), I upload the results into a floppy disk (this was, actually, my first time using a floppy disk. Nice to know that I'm learning how to use old technology as well as new technology.), so I can transfer them to a different computer that has a magical excel spreadsheet that makes sense of all the numbers. Again, I'll explain more about this later.

Tada! I found some results. And an old computer.

Okay, that was a mouthful! It was a good review for me, too. Somehow, I managed to replicate this process mostly by myself, and my results were good! (This was, of course, after I transferred the wrong data and accidentally got a flat pH of about 3. But you live and you learn.) So now I'm trusted to run actual experiments (because this was just to see if I could do it), and I have to do it with six vials. I had four vials this time: one blank without drug, one blank with drug, one wildtype M2 without drug, and one wildtype M2 with drug. I'll need two more so I can test drugs on a mutant strain of the virus. (And, since we have colored tape, I decided to make them rainbow colored to keep track of them. Because I could.)

Let me know if you have any questions, because I sure do (but this isn't really unusual).

Until next time!
- Lauren

P.S. After I wrote this, I learned that most of my concentrations and numbers were off. Thankfully, it's not my fault, since this is what I was told. But most of my numbers will change, although the process will remain the same, so I'm not going to edit what I've already written.



17 comments:

  1. Sounds like your adjusting well! How's dealing in μL? I work mostly with mLs, and even then my grip is a bit shaky. Hopefully it gets better with practice. Remember to catch the midnight train btw, it'll take you anywhere.

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    1. The pipettes are specialized for it, but my hand still shakes as I'm trying to put the pipette tip into the mixtures! At least the tips are plastic and harder to break.

      And yeah, that train goes on and on and on.

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  2. Lauren: Absolutely fascinating! You explanation of the process was very well written. It is exciting to me to hear how you are handling this process and equipment. I, for one, do not mind the detail that your describe your experiment in. You are definitely making the most of this opportunity.

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  3. Well finally you are using that jacket! How are my ear warmers? What pH are you suppose to be getting? And what actual experiments are you going to be doing if you know?

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    1. The pHs are usually between 2 and 3 of the mixture, from what I've been seeing, since I add HCl before starting the pH meter. My "actual" experiment is the exact same process, but I'll be testing an unknown drug rather than amantadine.

      And the ear warmers and gloves are great! I'm preparing for more cold weather this week.

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  4. Hi Lauren, I'm reading through and although it is interesting I do get overwhelmed. Sounds like you are doing great and progressing well. I did wonder though, why do you push the vial without the wildtype M2 channels through the filter 21 times? Does that make it legal?

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    1. Haha, I think it's legal before it's filtered anyways. All the liposomes are filtered. I thought 21 times was kind of arbitrary, but when I asked, apparently 21 is what they found worked best. Filtering it more doesn't seem to have an effect, and an odd number is needed so the mixture ends up in the left syringe when it starts in the right syringe.

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  5. Wow, floppy discs, huh? Who would've guessed. You mentioned making your process more efficient, what's a way you could do that?

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    1. There's a lot of down time during the process. For example, when I use the rotavapor, I have to wait anywhere from 5 to 15 minutes for all the fluid to be gone. I'm sure I should be doing something in this time (such as preparing more liposomes/lipids) but I haven't quite figured out what yet.

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  6. Lauren,
    Your work seems to me as an interesting idea and process to be doing. The humor in your posts make it really an easy read, and I felt as though it added your personality into your research project. It seems like this something that you are very excited about to learn more about. Your research seems very thorough, and you having error just helps you learn more and try to come up with a solution to fix it. I am Tanner from Lutheran High School in Parker, Colorado! We are in a class and our instructor toured your school, and s our assignment is to look over projects and these sound like so much fun.

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    1. Hi Tanner,

      Thank you so much for commenting on my blog! It's nice to know that people outside of my school are reading this as well. Do you think you'll be able to do a senior project also? Let me know if you have any other questions!

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    2. My instructor is thinking that we might, as there will five of us in our class, and he wants us to try it out. So hopefully we will be able to.

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    3. Wow, and I thought my class was small. How do you only have 5 people? Good luck for your project!

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  7. Hey Lauren, great update! I just came across a Windows 95 computer with a floppy drive at my lab...looks like these labs all have a bit of old tech hiding around. Also, just a question about the experiments: considering how high precision this work is, did it take you numerous tries to get it, or were you a natural at it?

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    1. Haha, I guess if the tech works, why replace it? (At least, that's what my uncle thinks.) I got results the first time, but that might be beginner's luck. The bigger test will be if i can replicate my results again. With any luck, I'll know tomorrow. Unfortunately, I might have left some of my lipids out of solution too long, so I don't know if I'll get anything this week.

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  8. That sounds like a long and complicated experiment. Did you spend the majority of your time actively experimenting, or was it mostly sitting around and waiting for stuff to finish?

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    1. Most of my time is spent cleaning materials and waiting. The actual experiment takes about ten minutes, while creating the liposomes takes a few days.

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