BacMam Actin and Lysoprobe Method Development in Human Embryonic Kidney and Mesenchymal Stem Cells

Jessica Adams, Amelia Ray, Dr. Heather Coan, Biology Department, Western Carolina University, 1 University Way, Cullowhee, NC 28723

A testing protocol was developed to confirm the proper operation of BacMam 2.0 probes Actin-RFP and Lysosome-GFP in human embryonic kidney (HEK) cells. Adult stem cells are useful in a variety of medical interventions in regrowing and repairing damaged tissue, but often lose efficacy when exposed to the stressful environment of damaged tissue. External studies have shown promising results that keratin can mitigate the stress stem cells experience by modifying the homeostatic cellular process of autophagy. Autophagy processes can be tracked via microscopy by transfecting cells with plasmids containing autophagy genes fused to fluorescent probes; however, stem cells do not readily respond to transfection, so methods to track autophagy in these cells are limited. In this study, we aimed to test a different type of system to determine whether BacMam probes can be successfully utilized in stem cells.  First, we tested HEK cells due to their low cost and ability to proliferate easily. HEK cells were seeded at 100,000 cells per well into 6-well tissue culture dishes. Cells were dosed with Actin-RFP and Lysosome-GFP BacMam probes and left overnight to incubate. Images were taken of the cells the next day with a Thermofisher EVOS fluorescent microscope. Preliminary results suggest the proper operation of the Lysosome-GFP BacMam probes in HEK cells, but there was no evidence of the Actin-RFP probe having worked properly. These results could be due to the cells being seeded at low density or too low of a probe dosage. Subsequent experiments are planned to optimize the probes in HEKs and then test them in adult stem cells. Pending promising results, future studies would move onto experimenting with inserting autophagy BacMam probes into adult stem cells in an effort to track the effects of keratin on autophagy and the resulting stress in adult stem cells.

Additional Abstract Information

Presenters: Jessica Adams, Amelia Ray

Institution: Western Carolina University

Type: Poster

Subject: Biology

Status: Approved

Time and Location

Session: Poster 2
Date/Time: Mon 3:00pm-4:00pm
Session Number: 2652