Serial Dilutions

Hey guys! Glad you could join me again! This week was a little all over the place, but I’m happy to know you guys are back here again! 

This week, I learned how to do serial dilutions, which are incredibly important in later parts of the experiment regarding treatment. Serial dilutions, as you might have guessed, is essentially a stepwise dilution. So first, I had to do some calculations to figure out how much milliliters or microliters to make with having some extra as well (just for safety purposes). I decided to go with 600 microliters, and I decided to do 8 dilutions (because the 96 well has 8 rows). 

Before we delve in, I wanted to go over my positive control, which is important later on in this story. I’m using SDS, which is sodium dodecyl sulfate, and it is known to basically kill all cells. Essentially, I’m using something that can kill cells to compare my combination treatment to. First, I pipetted 600 microliters of SDS into a small tube. Then for the next 6 small tubes, I pipetted 400 microliters of medium. Finally, for the last tube, I pipetted 600 microliters of medium. Then, I took 200 microliters from tube 1 and pipetted it into tube two, and resuspended it to mix it well, and then took 200 microliters of this new solution and pipetted it into tube three, and so forth. The last tube, however, remains only medium, while the first tube remains only SDS. 

Now that the serial dilution is complete, we can move forward with treatment. You guys have all seen the 96 well, and hopefully know it has rows and columns. So now, I will pipette 100 microliters per well of tube 1 into row 1, tube 2 into row 2, and so forth, until all the rows are completed. Then, I incubated the well for a day.

Now it’s time for the proliferation assay! The assay I am using is called the Alamar blue assay, and essentially it measures the mean fluorescence intensity (MFI). Alamar blue, or resazurin, is broken down by cells, and when it’s broken down, it exhibits fluorescence. Thus, it is a good measure for metabolic activity of cells, and this is what I’ll be using to measure whether the treatment has been working (less fluorescence = less metabolic activity = treatment working). First, I pipetted 20 microliter of Alamar blue into each well, and let it incubate for about an hour and a half. Then, I put the plate into the machine, which gave a reading of the MFI for each well. 

In the next update, I will explain how I will analyze this data and plot it, as well as go into details of making solutions for my treatment chemicals! That brings us to the end of today’s blurb, and I hope you enjoyed learning about serial dilutions and the actual assay! Until next time!