PhD Scientific Days 2024

Molecular Medicine I.

Improvement of Transient Agarose Spot (TAS) Cell Migration Assay: Introduction of Microplate-reader Based Detection and Evaluation

Text of the abstract

The scratch assay, the current gold standard method to investigate collective cell migration, a key cellular function involved in tumour invasion, wound healing or tissue fibrosis, has numerous limitations. Previously, our research group developed the similarly affordable, but more accurate Transient Agarose Spot (TAS) assay that was able to overcome most of the disadvantages, including the high intra and inter assay variability, however the detection and evaluation steps remained time consuming and equipment-intensive.
The aim of the study was to transfer the detection and evaluation process from graphical analysis of microscopic images into the more widespread microplate-reader, thereby making it suitable for high-throughput drug screening.
Cancer, fibroblast and epithelial adherent cell lines were seeded on 96-well plates containing agarose droplets. After removing the droplets various treatments were given to the cells to stimulate or inhibit their migration. The gap closure was followed for 72 hours using absorbance-based or fluorescent dyes and the scanning function of the microplate reader that determined the signal at 30 × 30 points in a 4 mm circle in each well.
Out of numerous dyes considering interfering factors, like the cytotoxicity, elimination and albumin binding, the fluorescent DNA staining Hoechst proved to be the most suitable, giving a stable fluorescent signal during the course of the experiment. The correlation between the gap areas determined by microscopy or microplate-reader was 93.5%, while the required hands-on time of the detection and evaluation decreased significantly.
In conclusion, the upgraded TAS assay provides a time- and cost-effective, accurate, reliable alternative to investigate cell migration that might even be applicable in the industrial field.
This research was founded by the National Research, Development and Innovation Office (NKFIH), K-142728; Semmelweis University, TKP2021-EGA-24, STIA-KFI-2021; Hungarian Research Network, ELKH-POC-2022-024, the New National Excellence Program of the Ministry for Culture and Innovation from the Source of the National Research, Development and Innovation Fund, ÚNKP-23-3-I-SE-36, ÚNKP-23-3-I-SE-42, ÚNKP-23-4-II-SE-29, ÚNKP-23-5-SE-15; Hungarian Academy of Sciences, János Bolyai Research Scholarship.

szasz.csenge@stud.semmelweis.hu
Semmelweis University
Dr. Veres-Székely Apor, PhD