Introducing Wound Healing Assays in the Undergraduate Biology Laboratory Using Ibidi Plates.

The wound healing assay is a simple and inexpensive method that allows researchers to experimentally mimic cell growth and migration leading to wound healing. In this assay, a wound is created on a monolayer of cultured mammalian cells and cell migration is monitored. Micrographs are captured at regular intervals during the duration of the experiment. These microscopy images are analyzed to compare cell migration and wound closure under different conditions. Introduction of different cytotoxic treatments into a wound healing assay can provide information as to whether a particular drug or compound of interest has the ability to affect cell migration. This type of analysis is important when assessing the ability of a particular cancer cell line to display invasive and metastatic behaviors. One of the challenges of this assay is to create the original wound in a way that is consistent across plates or treatments, facilitating comparisons across experimental groups. This is a particular challenge when using the wound healing assay in the context of an undergraduate biology class to expose students to a distinct form of mammalian cell culture and help them apply scientific knowledge and research skills. We found an easy way to overcome this obstacle by using ibidi plates. In this article, we provide a simple protocol to use ibidi plates and HeLa cells to set up wound healing assays. This laboratory exercise allows undergraduate students to utilize different skills developed through cell culture experience, such as growing, treating, and imaging mammalian cells.

YML018C protein localizes to the vacuolar membrane independently of Atg27p.

The function of the budding yeast YML018C protein remains to be determined. High-throughput studies have reported that the YML018C protein localizes to the vacuolar membrane and physically interacts with the autophagy-related protein Atg27p. While this evidence suggests a potential role for this uncharacterized protein in the process of autophagy, the function of this putative interaction remains uncharacterized. In this micropublication, we report our finding that the localization of the YML018C protein to the vacuolar membrane does not require Atg27p.

Using The Cancer Genome Atlas as an Inquiry Tool in the Undergraduate Classroom.

Undergraduate students in the biomedical sciences are often interested in future health-focused careers. This presents opportunities for instructors in genetics, molecular biology, and cancer biology to capture their attention using lab experiences built around clinically relevant data. As biomedical science in general becomes increasingly dependent on high-throughput data, well-established scientific databases such as The Cancer Genome Atlas (TCGA) have become publicly available tools for medically relevant inquiry. The best feature of this database is that it bridges the molecular features of cancer to human clinical outcomes-allowing students to see a direct connection between the molecular sciences and their future professions. We have developed and tested a learning module that leverages the power of TCGA datasets to engage students to use the data to generate and test hypotheses and to apply statistical tests to evaluate significance.

A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production.

The center of tumors, stem cell niches and mucosal surfaces all represent areas of the body that are reported to be anoxic. However, long-term study of anoxic cell physiology is hindered by the lack of a sustainable method permitting cell cultivation in the complete absence of oxygen. A novel methodology was developed that enabled anoxic cell cultivation (17d maximum time tested) and cell passage. In the absence of oxygen, cell morphology is significantly altered. All cells tested exhibited morphologic changes, i.e., a combination of tethered (monolayer-like) and runagate (suspension-like) morphologies. Both morphologies replicated (Vero and HeLa cells tested) and could be passaged anaerobically. In the absence of exogenous oxygen, anoxic cells produced reactive oxygen species (ROS). Anaerobic runagate HeLa and Vero cells increased ROS production from day 3 to day 10 by 2- and 3-fold, respectively. In contrast, anoxic tethered HeLa and Vero cells either showed no significant change in ROS production between days 3 and 10 or exhibited a 3-fold decrease in ROS, respectively. Detection of ROS was inversely related to detection of hypoxia-inducible factor-1α (HIF1) mRNA and HIF-1 protein expression which cycled over a 10-day period. This methodology has broad applications for the study of tumor and stem cell physiology as well as gastrointestinal cell-microbiome interactions. In addition, sustainable anaerobic cell culture may lead to the identification of novel pathways and targets for chemotherapeutic drug development.