Making stock solutions!

Hey guys! Welcome back to my blog! I hope you had a great spring break! 

Before we go into what I did this week, I promised to go over analyzing the data from last week, so here goes. Essentially, the proliferation assay gives us readings from each well of the mean fluorescent intensity – and we have 3 wells per each concentration of SDS in this case. So, we take the average and plot it, and then we also plot the standard deviation as the error bars. 

This is what the graph ended up looking like. For the most part, there is a trend that is quite visible – the lower the SDS concentration, the higher the MFI (which makes sense because it means that there are more cells that didn’t die). However, there are 2 weird points – which probably has to do with either contamination or pipetting errors. 

Onto this week’s work, which was all about stock solutions! What are they? They are concentrated solutions prepared ahead of time, and can be used to dilute and obtain solutions of desired concentrations. The compounds I’m using for the experiment, which are curcumin and quercetin, are in powder form, so it is necessary to make a stock solution out of them to be able to use it for treatments in my experiments. 

I parsed through a lot of scientific literature about these compounds, and I ended up finding that about 10-100μM was a good amount to use. However, to make a stock solution, it is always better to have a higher molar concentration than a lower molar concentration, so I decided to make a 100mM stock concentration for both curcumin and quercetin. 

So first, I found the molecular weight of the compounds through PubChem – curcumin was 368.39 g/mol and quercetin was 338.27 g/mol. So, 1 mM is equal to 0.368 mg/mL for curcumin and 0.338 mg/mL for quercetin, meaning that 100 mM = 36.84 mg/mL (curcumin) and 100mM = 33.83 mg/mL (quercetin). 

Now, I went ahead and measured out some curcumin and some quercetin on a scale. Since these were in high dosage powdered form, they were extremely toxic if inhaled. Therefore, I took the necessary precautions by wearing a face mask, a lab coat, and gloves. Since it was impossible to get it to 100 mg exactly, I just measured out some amount, and decided it would be easier to adjust the amount of DMSO to dissolve the compound and make it a 100mM solution instead of chasing the Herculean task of getting it to exactly 100 mg (which would also provide more sources of error). 

Here are my compounds after I measured them out.

To find the amount of DMSO needed, I divided the milligrams of the compound I measured  out by the 100mM value (36.84 mg/mL (curcumin) and 33.83 mg/mL (quercetin)). I ended up taking 155 mg of curcumin, so I needed 4.21 mL of DMSO for it, and I took 118 mg of quercetin, so I needed 3.49 mL of DMSO for it. 

I then mixed it up really well using a microliter pipette and ensured all of the compound was dissolved in the DMSO. However, because the compound was not under the fume hood during the measuring process, it is not sterile anymore, so I needed to make this new solution sterile again. For this, I used a sterilizing syringe, which essentially sucks up the solution using a syringe, and then passes it through a filter into a new bottle, thus sterilizing the solution. It was fairly simple to do, although I almost got poked by the needle 🙂. Now that the solutions were all made, I stored them in the fridge. 

I took this picture after I put them in the fridge for like an hour, and because of that the DMSO got a little frozen, which is why the solution don’t look solutiony.  

Anyway, I will be doing treatment with curcumin next week, (which I created a plan for and did all the calculations for this week) so that would mean that I needed a lot more cells to seed 6 96-well plates. Thus, I passaged my cells from a T25 flask (which holds 5 mL) to two T75 flasks (which both hold 18 mL each), so I have enough cells by Monday to start a round of the experiment. 

Stay tuned for next week, because that’s when things will get a lot more interesting!