RT Journal Article
SR Electronic
T1 Visualizing H<sub>2</sub>O<sub>2</sub> and NO in endothelial cells: strategies and pitfalls
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.02.15.528776
DO 10.1101/2023.02.15.528776
A1 Altun, Hamza Yusuf
A1 Secilmis, Melike
A1 Caglar, Tuba Akgul
A1 Vatandaslar, Emre
A1 Öztürk, Gürkan
A1 Vilain, Sven
A1 Eroglu, Emrah
YR 2023
UL http://biorxiv.org/content/early/2023/02/16/2023.02.15.528776.abstract
AB The relationship between hydrogen peroxide (H2O2) and nitric oxide (NO) in the vasculature is multifaceted and remains controversial because the dynamic detection of these reactive molecules is challenging. Genetically encoded biosensors (GEBs) allow visualizing real-time dynamics in living cells and permit multiparametric detection of several analytes. Although robust, GEBs’ utility depends on several parameters that need fine-tuning for proper imaging and correct data analysis: i.e., camera binning, temperature, and the resolution power of the imaging instruments are some critical parameters that require optimization. We have generated a new double-stable transgenic endothelial cell line stably expressing the biosensors HyPer7 and O-geNOp and systematically tested different imaging modes and their impact on the performance of each biosensor. Ambient temperature and the type of imaging mode did not influence the results, while camera resolution settings significantly affected readouts of HyPer probes but not O-geNOp. Changing a single parameter in a co-imaging mode significantly altered the biosensor’s dynamic measurements, potentially causing misinterpretation. This study provides a general guide and the pitfalls of employing GEBs in a multispectral imaging mode.Competing Interest StatementThe authors have declared no competing interest.