Week 9/10 – Scratching the Surface (literally and figuratively)

Over the past two weeks I have definitely felt everything slow down, contrary to what last week’s blog post title would imply haha. The end is very near and I am not rushing to discover something entirely original with my research. That is not to say there is nothing more to accomplish in the coming weeks. I am going to do one or two more experiments and then present everything I have learned! I am not entirely sure how I am going to summarize everything and form a conclusion, but there is still time to figure that out ūüôā

I am not going to share any of the results from experiments done over Week 9 and 10 in this blog post, because I rather share information about a new experiment and keep this post short.

To summarize Week 9: I finished the proliferation assay with 19-26 and¬†RvD1 and analyzed the results. The results are quite interesting and I will share more next week. I corrected the growth curve assay results using a standard curve, as my external advisor suggested. I also¬†followed one lab member’s exosome experiment and had the opportunity to do some of the steps!¬†

This week I learned a new experiment called a scratch assay. Unlike a proliferation assay, which measures the proliferation rate of vascular smooth muscle cell, a scratch assay focuses on the migration rate of these cells.

A scratch assay involves creating a “scratch” on a cell monolayer. A pipet tip is used to draw a horizontal line through each well to create a scratch. Images are captured with a fancy microscope at the beginning (when the scratch is made) and the end (after about 16 hrs.). A formula is used to calculate the % of would closure in every well. The formula is the area of the scratch at the beginning – the area of the scratch at the end / the area at the beginning.

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Creating this tiny line in a circular well was way harder than it looked. Most of my lines were not straight this time, but many of the lab members gave me tips for the future.

Within the context of my project, a scratch assay will be used to determine the effects of 19-26 and RvD1 on the migration of smooth muscle cells. The assay helps mimic cell migration during wound healing. If you remember back to Week 4, I explained that in the inflammation process vascular smooth muscle cells migrate from the tunica media to the tunica intimia to form the neointima. The goal is determine which drug minimizes this migration the most as the formation of the neoinitima is essentially restenosis.

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My cell plate had 4 wells (quad-duplicates) with a negative control (no treatment to the cells), 4 wells with the positive control PDGF (read more about PDGF here: Week 7), 4 wells with RvD1 at 10 nM, 12 wells with 19-26 at 1, 10, and 100 nM concentrations (4 wells per concentration).

A positive control is a group in which a response is expected. It controls for unknown variables. We want to make sure that the cells are migrating/not migrating only because of the drug treatment. The positive control has to be something that will close the wound (or cause cells to migrate). PDGF is this control as it regulates cell migration. Every single well in the assay will also have PDGF as we want to see the results in spite of the positive control’s response.

The results were surprising to both me and the lab member helping me. We were both shocked to see that 19-26 at a 100nM concentration completely prohibits migration (which is impossible, even for a “miracle” drug) to the point where the cells were dying. This raises the question whether 100nM of the drug is extremely toxic, which can be confirmed by the cytotoxicity assay results from Week 8.

Here are some pictures from the assay using the fancy microscope. I used these pictures and a software called ImageJ to calculate the % wound closure. This is what the negative control well (no treatment) looked like:

Screen Shot 2018-04-20 at 3.43.48 PM.png

0 hrs.                                                                                               16 hrs.

As you can see, the scratch is the gap in the middle at 0 hrs. After 16 hrs, some of the cells migrated to fill gap. And here are the picture from the 100nM 19-26 drug treatment:

Screen Shot 2018-04-20 at 3.48.35 PM

0 hrs.                                                                                               16 hrs.

Here, the area of the scratch at the beginning and the end look about the same, which implies that almost none of the cells migrated. You can also notice that cells look shriveled and small in the 16 hr image, the drug treatment was surely too toxic. And here is the effects of 19-26 at 1nM, for comparison sake:

Screen Shot 2018-04-20 at 3.52.51 PM.png

0 hrs.                                                                                               16 hrs.

All of the questions the results from these experiments are bringing up makes me feel as if I have only scratched the surface. But over 12 weeks only so much is possible. To reach a more reliable conclusion from the data with minimized error these experiments would have to replicated multiple times. Possibly a task for the far off future! AS_60903_L

This week I also had the chance to do some of the data analysis for other experiments people are doing in the lab. Since I have no idea what the results should look like for their experiments they feel my analysis will have no bias. I am learning a lot about how to use the fancy microscope and graphing data from this opportunity as well.

The American Heart Association has named April the Move More Month! Read more about this here: April is Move More Month

Therefore, this week’s risk factors: Physical Inactivity and Obesity

Physical activity is crucial for prevention of coronary heart disease. Regular, moderately vigorous physical activity helps reduce the risk of blood vessel clogging. As I said in Week 8, it can be as easy as fulfilling the recommended 30 minutes of moderate-intensity aerobic activity at least 5 days per week. Exercise is the solution to many risk factors associated with this disease. High blood pressure from two weeks ago, Screen-Shot-2018-02-08-at-10.10.11-PMdiabetes from the week before, and obesity this week can all be controlled with regular physical activity. Studies have found that individuals that are overweight are more likely to develop heart disease. They are more at risk for Type 2 diabetes and the many other risk factors of cardiovascular disease.

615b3-14nixsi.gifTo learn more about how to get active and manage your weight: Easy Tips to Get Moving, Controlling Obesity

Hope you will spend the rest of April being active and join me next week for the second to last blog post ūüôā

2 thoughts on “Week 9/10 – Scratching the Surface (literally and figuratively)

  1. Hey Amrita,

    I can see that your knowledge on this subject has grown tremendously. I am just curious what you think is the most valuable piece on information you have learned from this experience and any difficulties you went through (like whether you regretted making certain decisions that you might have changed if you went through this experience all over again). I’m so proud of you Amrita! Looking forward to reading your upcoming posts ūüôā

    Liked by 1 person

    • It is hard to pick one piece of information. The research techniques I learned were the most valuable though. There were also a lot of difficulties I experienced, but I don’t regret anything and wouldn’t change a thing! I had a lot of trouble with data analysis and calculations, as well as the scratch assay technique. And overall the biggest issue I had was having to stray away from my initial proposal. Thank you Manu!

      Like

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