Over the course of the summer, the research I worked on was understanding the role of turicibacter and butyrate in intestinal tumorigenesis. The scope of this project was to observe the effects of butyrate, an intestinal microbial metabolite of dietary fiber that exhibits chemoprevention effects of colorectal cancer, the third most commonly diagnosed cancer and the third leading cause of cancer-related deaths in the United States for both men and women. Turicibacter is a butyrate-producing bacteria found in the gut of animals (reduced amounts in obese animals) in which scientists still know very little about.
To examine the effects of butyrate, we treated Caco-2 cells (colorectal cancer cells) with varying concentrations of butyrate. I started off by using a general cell culturing protocol in which Caco-2 cells were maintained in 100mm petri dishes in a 37°C incubator with a humidified atmosphere of 5% CO2. The cells’ monolayers were grown in cell culture media, freezing media, and 0.25% Trypsin-EDTA solution. The culture medium was replaced daily and rinsed with 1 mL of PBS per 100 mm dish twice to aspirate the supernatants out. The Caco-2 cells were seeded on a 96-well plate and cultured for 48 hours. The 96-well plates were replaced with fresh medium and different concentrations of sodium butyrate (1, 2, and 5 mM) and were further incubated for 72 hours. By the end of the treatment, the wells were added with 50 μL of a 1 mg/mL solution of MTT in PBS and were incubated for another 4 hours at 37°C. We discarded the supernatant and the colored formazan crystals were solubilized with 100 μL/well of DMSO. We proceeded to measure the absorbance of the 96-well plate with a microplate spectrophotometer at 570 nm. Though I was not able to finish the project in the span of three months, the basic cell culturing protocol and MTT assay has helped me prepare for future laboratory work and I look forward to continuing this project in the upcoming months.
