April 3rd - 7th
This week, I focused on staining and imaging slides with iron-labelled 231 BR cells. I was able to image two slides, containing 200,000 iron-labelled 231 BR cells. One slide contained cells labelled with a labelling concentration of 50 ug/mL of ProMag, and another slide contained cells labelled with a concentration of 75 ug/mL of ProMag.
For the imaging, our lab uses two stains to visualize the iron-labelled cells. One stain, called pearls' prussian blue (PPB) stains the iron blue. The other stain, nuclear fast red, is a counterstain which stains each fixed cell nuclei red. This reveals the location of each cell relative to the iron. With this combination of staining, one can visualize whether the iron was taken up by each cell, and therefore how effective the cell labelling was.
This figure was taken from Knier et al., 2020. (http://dx.doi.org/10.1007/s10585-020-10044-0) Panel A shows an example of PPB staining with MDA-MB-231 BR cells. The pink/red colour is the nuclear fast red stain, and the blue colour is the PPB stain.
For my reference, I am browsing images like this and including them in my blog. This will help me better understand what a successful PPB staining experiment should look like. Notice that in the image, most of the iron is intracellular, and there is little iron found in the extracellular space. This is indicative of effective iron labelling. For this reason, PPB staining and imaging is used to assess the labelling efficiency.
In my experiment, I conducted labelling with three different loading concentrations of iron, and I wanted to assess which concentration would work best. Unfortunately, my nuclear fast red stain did not work this week. Although I was able to see where the iron was located, I couldn't tell whether the iron was within cells or in the extracellular environment. This means that I will need to repeat the experiment next week to optimize the loading concentration of ProMag that will be used for the comparison with BLI. Luckily, I had saved cell pellets with each of the three labelling concentrations. Next week, I will redo the PPB staining using these samples and hopefully get clearer images.
My main takeaway from this week is that mistakes will inevitably happen in the lab, however I can do my best to be efficient and minimize avoidable mistakes. My goal for next week is to practice critical thinking while performing each step of an experimental protocol. This involves reminding myself of how certain reagents work as I add them, or why I am using certain dyes in my cell staining and imaging protocol. I believe that this simple act of being fully present while running an experimental protocol will decrease my margin of mistakes made in the lab, and increase my efficiency and productivity. This will greatly benefit me in the transition to grad school this coming September.