Week 6 – Taking a Step Back

This week, I decided to step back and conduct a foundational experiment that will reveal more about the nature of vascular smooth muscle cells. My external advisor suggested that I create a base line with a growth assay. The end result of this assay will be multiple growth curves of smooth muscle cells’ response to different amounts of nutrients given. The goal is to be able to compare this assay to the resolvin assay and future assays. The MTT assay with RvD1 is now planned for Week 8. Essentially, I am first establishing a stronger control group to compare the treated RvD1 cells to. I mentioned in Week 2/3 that I would do an assay with no drug treatment and this is exactly that.

A quantitative cell proliferation assay (growth assay) is way to measure cell proliferation in response to stimuli.  In the pervious experiment this stimuli was RvD1. This time it is different concentrations of serum. The protocol is extremely similar to the AlamarBlue assay: Alamarblue Proliferation Assay (Serum) Protocol

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Incubator kept at 37 C: home to the cells

I used the same vascular smooth muscle cells from a saphenous vein and seeded approximately 800 cells per well. I treated the cells with different concentrations of serum, added AlamarBlue, read the cell number, added more serum, incubated, and repeated every other day. I just finished Day 3 of this assay and will continue until about Day 11. As you can see in the cell plate below, I am comparing .5% FBS, 1% FBS, and 10% FBS. I also have a serum-free media and Vitamin C control, same as last week.

Serum, specifically fetal bovine serum (FBS), is used when culturing cells because it contains a low level of antibodies, essential growth factors, and controls pH changes. Serum is added to the media (another liquid to support the growth of cells).

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The actual cell plate 🙂

I am still analyzing the results from this week and will let the cells continue to grow through next week. What should the results look like? Elevated levels of serum should result in more availability of nutrients for the cells. Thus higher levels of serum will promote a higher percentage of cells to undergo cell division. This translates to: the wells with 10% FBS should have more cell growth. My external advisor also said if everything runs smoothly the growth curve should be exponential.

I have come to the realization, with the help of Dr. Murthy, that my intended proposal may have been a stretch and that quantitatively comparing the results of drugs in the lab is not practical (cost, time, and experience wise). I am instead reading about newer drugs used in stents and hope to share this information qualitatively. Through the experiments in the lab I am learning a lot about vascular smooth muscle cell growth and responsiveness, which can be used as stepping stone to explore bigger questions concerning restenosis and drugs in drug-eluting stents. I am also going to be focusing more on the effects of resolvin, rather than the 3 drugs I initially intended, on the cells.

With this in mind, the plan for next week includes starting a cytotoxicity assay and continuing the growth assay. Both these assays will reveal more about how these cells react to different conditions, such as drugs or resolvin.

A cytotoxicity experiment will measure a decrease in cell proliferation in response to a toxic compound. When cells are exposed to a cytotoxic compound they undergo necrosis or apoptosis. If a compound is found to be toxic scientists know to eliminate it or dilute its concentration. I will be measuring the toxicity of RvD1 in order to continue measuring this resolvin’s effectiveness.

This week’s risk factor: High Blood Pressure

High blood pressure adds to the heart’s workload, causing the heart muscle and arteries to thicken and become stiff. This stiffening narrows the passage through which blood can flow to the heart, exacerbating the problem of restenosis even after CRO_Health_High_Blood_Pressure_Chart_11-14a stent is inserted. It also increases your risk of stroke, heart attack, and congestive heart failure. You can make lifestyle changes (limit alcohol, intake less salt, and exercise more) and regularly monitor your blood pressure to steer clear of damage to your heart.

To learn more about how you can control your blood pressure: Changes You Can Make to Manage High Blood Pressure

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See you next week! 🙂

 

3 thoughts on “Week 6 – Taking a Step Back

  1. Hi Amrita

    Your blog is very informative and I am learning a lot about drug eluting stents.You are doing a great job! Look forward to reading the next week blog!

    Liked by 1 person

  2. Pingback: Week 7 – Growth Curves and a New Drug | The Effectiveness of Drug-Eluting Stents

  3. Pingback: Week 8 – Much More to Come | The Effectiveness of Drug-Eluting Stents

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